Recombinant herpes simplex virus 2 (HSV-2) vaccine vectors

ABSTRACT

Recombinant herpes simplex virus 2 (HSV-2) vaccine vectors, virions thereof, compositions and vaccines comprising such, and methods of use thereof are each provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT InternationalApplication PCT/US2015/018272, filed Mar. 2, 2015, which claims benefitof U.S. Provisional Application No. 61/946,965, filed Mar. 3, 2014, andof U.S. Provisional Application No. 62/080,663, filed Nov. 17, 2014, thecontents of all of which are hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersAI061679, AI51519, AI097548, AI026170 and AI065309 awarded by theNational Institutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to,including by number in square brackets. Full citations for thesereferences may be found at the end of the specification. The disclosuresof these publications, and all patents, patent application publicationsand books referred to herein, are hereby incorporated by reference intheir entirety into the subject application to more fully describe theart to which the subject invention pertains.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) persist assignificant health problems globally, disproportionally impactingdeveloping countries and poor communities around the world and fuelingthe HIV epidemic. Vaccines are urgently needed for these infections ascurrently there is no effective vaccine for HSV-1, HSV-2 or HIV. HSV-1is the primary cause of infectious blindness, while HSV-2 is the primarycause of genital ulcers globally, although HSV-1 is now more commonlyidentified in association with genital tract disease in developedcountries. Genital herpes is a recurrent, lifelong disease that canstigmatize and psychologically impacts those affected. Infection withHSV-2 significantly increases the likelihood of acquiring andtransmitting HIV, while vertical transmission of either serotype oftenleads to severe infant morbidity or death. Recent clinical trials ofHSV-2 vaccines based on sub-unit formulations using viral glycoproteinsD alone or in combination with glycoprotein B (gD and gB) have failed,despite inducing systemic neutralizing antibodies. Surprisingly an HSV-2gD subunit (gD-2) vaccine provided partial protection against HSV-1, butno protection against HSV-2. Several attenuated viruses been evaluatedpre-clinically, but clinical studies to date have been limited totherapeutic applications (reducing frequency of recurrences) and havealso failed to show efficacy. Thus, novel vaccine strategies must beengineered and evaluated.

The present invention addresses this need for new and improved HSV-1 andHSV-2 vaccines.

SUMMARY OF THE INVENTION

An isolated, recombinant herpes simplex virus-2 (HSV-2) is providedhaving a deletion of an HSV-2 glycoprotein D-encoding gene (U_(s6)) inthe genome thereof.

Also provided is a virion of an isolated, recombinant HSV-2 having adeletion of an HSV-2 glycoprotein D-encoding gene (U_(s6)) in the genomethereof.

An isolated cell is provided comprising therein a recombinant HSV-2genome as described herein or a recombinant HSV-1 gene as describedherein, wherein the cell is not present in a human being.

Also provided is a vaccine composition comprising the recombinant HSV-2virus as described herein, or the virion as described herein.

Also provided is a composition comprising the recombinant HSV-2 virus asdescribed herein, or the virion as described herein, wherein the genomeof the virus or virion comprises at least a deletion of a second gene,wherein the second gene is necessary for HSV-2 viral replication orvirulence.

A pharmaceutical composition comprising the recombinant HSV-2 virus asdescribed herein, or the virion as described herein, and apharmaceutically acceptable carrier.

Also provided is a method of eliciting an immune response in a subjectcomprising administering to the subject an amount of (i) the recombinantHSV-2 virus as described herein; (ii) a virion thereof as describedherein, (iii) the vaccine as described herein; (iv) a composition asdescribed herein; or (v) a pharmaceutical composition as describedherein, in an amount effective to elicit an immune response in asubject.

Also provided is a method of treating an HSV-1, HSV-2 or HSV-1 and HSV-2co-infection in a subject or treating a disease caused by an HSV-1,HSV-2 or co-infection in a subject comprising administering to thesubject an amount of (i) the recombinant HSV-2 virus as describedherein; (ii) a virion thereof as described herein, (iii) the vaccine asdescribed herein; (iv) a composition as described herein; or (v) apharmaceutical composition as described herein, in an amount effectiveto treat an HSV-1, HSV-2 or co-infection or treat a disease caused by anHSV-1, HSV-2 or co-infection in a subject.

Also provided is a method of vaccinating a subject for HSV-1, HSV-2 orco-infection comprising administering to the subject an amount of (i)the recombinant HSV-2 virus as described herein; (ii) a virion thereofas described herein, (iii) the vaccine as described herein; (iv) acomposition as described herein; or (v) a pharmaceutical composition asdescribed herein, in an amount effective to vaccinate a subject forHSV-1, HSV-2 or co-infection.

Also provided is a method of immunizing a subject against HSV-1, HSV-2or co-infection comprising administering to the subject an amount of (i)the recombinant HSV-2 virus as described herein; (ii) a virion thereofas described herein, (iii) the vaccine as described herein; (iv) acomposition as described herein; or (v) a pharmaceutical composition asdescribed herein, in an amount effective to immunize a subject againstHSV-1, HSV-2 or co-infection.

In an embodiment of the vaccines, compositions and pharmaceuticalcompositions, and of the methods of use thereof, the amount ofrecombinant HSV-2 is an amount of pfu of recombinant HSV-2 effective toachieve the stated aim.

Also provided is a method of producing a virion of a recombinant herpessimplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoproteinD-encoding gene in the genome thereof and comprising a HSV-1 or HSV-2glycoprotein D on a lipid bilayer thereof, comprising infecting a cellcomprising a heterologous nucleic acid encoding a HSV-1 or HSV-2glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) havinga deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof under conditions permitting replication of the recombinantherpes simplex virus-2 (HSV-2) and recovering a HSV-2 virion produced bythe cells.

Also provided is a recombinant nucleic acid having the same sequence asa genome of a wild-type HSV-2 except that the recombinant nucleic aciddoes not comprise a sequence encoding an HSV-2 glycoprotein D.

Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2)having a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof for treating or preventing an HSV-1, HSV-2 or co-infection in asubject.

Also provided is a virion of an isolated, recombinant HSV-2 having adeletion of an HSV-2 glycoprotein D-encoding gene in the genome thereoffor treating or preventing an HSV-1, HSV-2 or co-infection in a subject.

An isolated, recombinant herpes simplex virus-2 (HSV-2) is providedhaving a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof.

Also provided is a virion of an isolated, recombinant HSV-2 having adeletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof.

Also provided is an isolated cell comprising therein a virus asdescribed herein or a virion as described herein, wherein the cell isnot present in a human being.

A vaccine composition comprising a virus as described herein, or avirion as described herein.

Also provided is a composition comprising a virus as described herein,or a virion as described herein, wherein the genome of the virus orvirion comprises at least a deletion of a second gene, wherein thesecond gene is necessary for HSV-2 viral replication.

Also provided is pharmaceutical composition comprising a virus asdescribed herein, or a virion as described herein, and apharmaceutically acceptable carrier.

Also provided is a method of eliciting an immune response in a subjectcomprising administering to the subject an amount of (i) a virus asdescribed herein; (ii) a virion as described herein, (iii) a vaccine asdescribed herein; (iv) a composition as described herein; or (v) apharmaceutical composition as described herein, in an amount effectiveto elicit an immune response in a subject.

Also provided is a method of treating an HSV-2 infection in a subject ortreating a disease caused by an HSV-2 infection in a subject comprisingadministering to the subject an amount of (i) a virus as describedherein; (ii) a virion as described herein, (iii) a vaccine as describedherein; (iv) a composition as described herein; or (v) a pharmaceuticalcomposition as described herein, in an amount effective to treat anHSV-2 infection or treat a disease caused by an HSV-2 infection in asubject.

Also provided is a method of vaccinating a subject for HSV-2 infectioncomprising administering to the subject an amount of (i) a virus asdescribed herein; (ii) a virion as described herein, (iii) a vaccine asdescribed herein; (iv) a composition as described herein; or (v) apharmaceutical composition as described herein, in an amount effectiveto vaccinate a subject for HSV-2.

Also provided is a method of immunizing a subject against HSV-2infection comprising administering to the subject an amount of (i) avirus as described herein; (ii) a virion as described herein, (iii) avaccine as described herein; (iv) a composition as described herein; or(v) a pharmaceutical composition as described herein, in an amounteffective to immunize a subject against HSV-2.

Also provided is a method of producing a virion of a recombinant herpessimplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoproteinD-encoding gene in the genome thereof and comprising an HSV-1glycoprotein D on a lipid bilayer thereof, comprising infecting a cellcomprising a heterologous nucleic acid encoding a HSV-1 glycoprotein Dwith a recombinant herpes simplex virus-2 (HSV-2) having a deletion ofan HSV-2 glycoprotein D-encoding gene in the genome thereof underconditions permitting replication of the recombinant herpes simplexvirus-2 (HSV-2) and recovering a recombinant HSV-2 virion comprising anHSV-1 glycoprotein D on a lipid bilayer thereof produced by the cell.

Also provided is a method of producing a virion of a recombinant herpessimplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoproteinD-encoding gene in the genome thereof and comprising a non-HSV-2 surfaceglycoprotein on a lipid bilayer thereof, comprising infecting a cellcomprising a heterologous nucleic acid encoding the non-HSV-2 surfaceglycoprotein with a recombinant herpes simplex virus-2 (HSV-2) having adeletion of an HSV-2 glycoprotein D-encoding gene in the genome thereofunder conditions permitting replication of the recombinant herpessimplex virus-2 (HSV-2) and recovering a recombinant HSV-2 virioncomprising a non-HSV-2 surface glycoprotein on a lipid bilayer thereofproduced by the cell.

Also provided is a recombinant nucleic acid is provided having the samesequence as a genome of a HSV-2 except that the sequence does notcomprise a sequence encoding an HSV-2 glycoprotein D.

Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2)having a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof for treating or preventing an HSV-2 infection in a subject.

Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2)having a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof for treating or preventing an HSV-1 infection in a subject.

Also provided is a virion of an isolated, recombinant HSV-2 having adeletion of an HSV-2 glycoprotein D-encoding gene in the genome thereoffor treating or preventing an HSV-2 infection in a subject.

Also provided is a method of treating an HSV-1 infection, or HSV-1 andHSV-2 co-infection, in a subject, or treating a disease caused by anHSV-2 infection or HSV-1 and HSV-2 co-infection in a subject comprisingadministering to the subject an amount of (i) a virus as describedherein; (ii) a virion as described herein, (iii) a vaccine as describedherein; (iv) a composition as described herein; or (v) a pharmaceuticalcomposition as described herein, in an amount effective to treat anHSV-2 infection or treat a disease caused by an HSV-2 infection in asubject or an amount effective to treat an HSV-1 and HSV-2 co-infectionor treat a disease caused by an HSV-1 and HSV-2 co-infection in asubject.

Also provided is a method of vaccinating a subject for an HSV-1infection, or HSV-1 and HSV-2 co-infection, comprising administering tothe subject an amount of (i) a virus as described herein; (ii) a virionas described herein, (iii) a vaccine as described herein; (iv) acomposition as described herein; or (v) a pharmaceutical composition asdescribed herein, in an amount effective to vaccinate a subject for anHSV-1 infection, or HSV-1 and HSV-2 co-infection.

Also provided is a method of immunizing a subject against an HSV-1infection, or HSV-1 and HSV-2 co-infection, comprising administering tothe subject an amount of (i) a virus as described herein; (ii) a virionas described herein, (iii) a vaccine as described herein; (iv) acomposition as described herein; or (v) a pharmaceutical composition asdescribed herein, in an amount effective to immunize a subject againstan HSV-1 infection, or HSV-1 and HSV-2 co-infection.

Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2)having a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof and further comprising a heterogenous antigen of a pathogen.

Also provided is a method of inducing antibody dependent cell mediatedcytotoxicity (ADCC) against an antigenic target in a subject comprisingadministering to the subject an isolated, recombinant herpes simplexvirus-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encodinggene in the genome thereof and further comprising a heterogenous antigenon a lipid bilayer thereof in an amount effective to induce antibodydependent cell mediated cytotoxicity (ADCC) against an antigenic target.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: HSV-2 ΔgD initiates an abortive infection: HSV-2 ΔgD−/+ onlyreplicates successfully in cells that provide gD in trans (e.g. VD60[40, 41]), but not in cells such as Vero cells (ATCC CCL-81, Greenmonkey kidney) or CaSki (ATCC CRL-1550, Homo sapiens, cervix) that donot encode U_(S)6. Non-complemented HSV-2 ΔgD (ΔgD−/− obtained from Verocells) cannot infect cells such as Vero and CaSki, which do not encodeU_(S6).

FIG. 2A-C: A. Severe combined immunodeficiency (SCID) mice inoculatedwith up to 10⁷ plaque-forming units (pfu) of HSV-2 ΔgD−/+ virus do notmanifest signs of disease after high dose intravaginal or subcutaneousinoculation. In contrast SCID mice inoculated with wild-type virus at a1,000-fold lower viral dose (10⁴ pfu) succumb to disease. Survivalcurves are shown in A, epithelial scores (scale of 0 to 5) for evidenceof erythema, edema, or genital ulcers in B and neurological scores(scale of 0 to 5) for evidence of neuronal infection in C.

FIG. 3A-C: Immunization with HSV-2 ΔgD−/+ virus elicits anti-HSV-2antibodies. While sc.-sc. immunization elicits significant levels ofboth systemic and mucosal (vaginal washes) anti-HSV-2 antibodies,sc.-i.vag. immunization with HSV-2 ΔgD−/+ elicits lower levels ofsystemic anti-HSV-2 antibodies and no increase in antibody levels invaginal washes. Anti-HSV-2 antibody levels in serum are shown in A andanti-HSV-2 antibody levels in vaginal washes are shown in B. Miceimmunized with ΔgD−/+ display neutralizing anti-HSV-2 antibodies in theserum after challenge with virulent HSV-2. The neutralizing capacity ofthe antibodies elicited by ΔgD−/+ immunization is shown in C. (* p<0.05;**p<0.01; ***p<0.001).

FIG. 4A-C: A: CD8+ gBT-I T cell counts in spleens of C57Bl/6 micetransferred with Tg T cells, then primed and boosted with HSV-2 ΔgD−/+or VD60 lysate (Control). B: Percentage of gBT-I memory T cells inspleens of vaccinated or Control mice. C: 14 days after boost,splenocytes were isolated and re-stimulated in vitro with gB498-505peptide and analyzed 6 hr later for cytokine production by intracellularcytokine staining and flow cytometry. (*p<0.05; **p<0.01; ***p<0.001).

FIG. 5A-F: Immunization with HSV-2 ΔgD−/+(10⁶ pfu/mouse) protects micefrom a lethal HSV-2 challenge. Mice were primed subcutaneously andboosted 3-weeks apart either sc. or i.vag. and then challenged 3-weeksafter boost intravaginally with an LD₉₀ of virulent wild-typeHSV-2(4674). While Control (immunized with the VD60 cell lysate) micesuccumbed to disease, as manifested by significant weight loss (A) anddeath (B), ΔgD−/+-immunized mice displayed significantly less pathology.Furthermore, ΔgD−/+-immunized mice showed less epithelial disease (C)and neurological pathology (D) after lethal challenge. Additionally,ΔgD−/+-vaccinated mice displayed significantly less viral loads invaginal washes (E), vaginal tissue and dorsal root ganglia (DRG) (F)after intravaginal challenge with a lethal dose of virulent HSV-2compared to mice immunized with VD60 cell lysate as a Control. Noinfectious virus could be recovered from ΔgD−/+-immunized mice in Day 4vaginal washes or Day 5 vaginal tissue and DRG. (*p<0.05; **p<0.01;***p<0.001).

FIG. 6A-C: Mice immunized with HSV-2 ΔgD−/+ secrete less inflammatorycytokines in vaginal washes after challenge with virulent HSV-2. Miceimmunized with HSV-2 ΔgD−/+ secrete less TNF-α, IL-6 and IL-1β invaginal washes than mice immunized with VD60 lysate and challenged withvirulent HSV-2. Differences in inflammatory cytokine expression areobserved at different time-points after challenge. (*p<0.05; **p<0.01;***p<0.001).

FIG. 7A-D: Immunization with HSV-2 ΔgD−/+ recruits T cells to theinfection site and associated LNs. Mice immunized sc.-sc. with ΔgD−/+displayed increased percentages of activated anti-HSV-2 gBT-I CD8+ (A)and CD4+ T cells (B) in sacral lymph nodes (LNs) after challenge withvirulent HSV-2. LNs were extracted and incubated 6 h with UV-inactivatedΔgD−/− and then stained with antibodies for flow cytometry analysis.Mice immunized sc.-i.vag. with ΔgD−/+ displayed increased numbers ofanti-HSV-2 gBT-I CD8+ (C) and CD4+ T cells (D) in the vagina afterchallenge with virulent HSV-2. Vaginal tissues were processed to extractT cells and stained with antibodies for flow cytometry analysis. Cellcounting was done with (CountBright™, Lifetechnologies). (*p<0.05;**p<0.01).

FIG. 8A-8C. HSV-2 ΔgD-2 provides complete protection against diseasefollowing intravaginal or skin challenge with vaccine doses as low as5×10⁴ PFU. C57BL/6 mice were primed and then 21 days later boostedsubcutaneously (sc) with either 5×10⁴ PFU, 5×10⁵ PFU, 5×10⁶ PFU ofHSV-2ΔgD-2 or VD60 lysates (control). Mice were subsequently challenged21 days after boost with an LD90 of HSV-2(4674) either (8A)intravaginally or (8B) via skin scarification and followed for survival(n=5 mice/group) and disease scores. (8C) Serum was assessed for HSV-2antibodies before (PreBleed), day 7 post-prime, and day 7 post boost viaELISA (line represents mean). *p<0.05, **p<0.01, ***p<0.001, ΔgD-2vaccinated groups vs. control-vaccinated group via two-way ANOVA. KaplanMeier analysis was used for survival curves.

FIG. 9A-9D. Mice vaccinated with HSV-2 ΔgD-2 are protected againstclinical isolates of HSV-1 and HSV-2. C57BL/6 (n=7 mice/group) or Balb/C(n=5 mice/group) mice were immunized with ΔgD-2 or VD60 cell lysates(Control) and subsequently challenged with an LD90 dose of most virulentisolates and monitored daily for lesions in the skin (9A; representativeimages from C57BL/6 mice), survival (9B; C57BL/6 mice and 9C; Balb/C)Additional C57BL/6 mice were challenged with 10 and 100 times (10× and100×) the LD90 dose of SD90 and 10× the LD90 of Bx³1.1 and monitored forsurvival (9D). Survival for HSV-2 ΔgD-2-vaccinated group vs.control-vaccinated group were compared by Kaplan Meier analysis,***p<0.001).

FIG. 10A-10D. Virus is rapidly cleared and no latent virus is detectedin HSV-2 ΔgD-2 immunized mice following challenge with clinicalisolates. Mice were immunized with ΔgD-2 or VD60 cell lysates (Control)and subsequently challenged by skin scarification with an 1× or 10× theLD90 of HSV-1(B³×1.1) or with 1×, 10× or 100× the LD90 of HSV-2(SD90)(n=5 mice per group). Skin biopsies were obtained on day 2 and day 5post-challenge and assayed for viral load by plaque assay on Vero cells(10A) (n=3 samples/group, line represents mean). The presence ofreplicating or latent HSV in DRG tissue obtained from ΔgD-2 vaccinated(day 14 post challenge) or control vaccinated (time of euthanasia) miceby plaque assay (10B) and qRT-PCR (10C), respectively (n=5 mice/group).Latency was further evaluated by co-culturing Vero cells with DRGisolated from ΔgD-2 and control immunized mice that were challenged withan LD90 of HSV-2 SD90 at day 5 post-challenge (10D). Data in Panels Band C are presented as box and whisker plots with black dots indicatingoutliers. HSV-2 ΔgD-2-vaccinated group and control-vaccinated groupswere compared by student's t-test; *p<0.05; **p<0.01; ***p<0.001.

FIG. 11A-11D. HSV-2 IgG2 specific antibodies are rapidly recruited intothe skin of HSV-2 ΔgD-2 vaccinated mice following viral challenge. (11A)Mice were immunized with ΔgD-2 or VD60 cell lysates (Control) andsubsequently challenged with HSV-1(B³×1.1) and HSV-2(SD90) clinicalisolates on the skin. Skin biopsies were obtained 21 days post-boost andday 2 post-challenge and evaluated for the presence of anti-HSVantibodies in homogenates (1:10³ dilution) by ELISA using an HSV-2(left) or HSV-1 (right) infected cell lysate as the antigen (n=3 miceper group, line represents mean). To further quantify the HSV-specificantibodies in the skin, pools of skin homogenates were serially dilutedand assayed in the HSV-2 ELISA (6 mice per pool and results are mean±SDobtained from duplicates) (11B). The ratio of anti-HSV-2 IgGsub-isotypes in the day 2 post-challenge skin homogenate pool wasdetermined using sub-isotype specific secondary antibodies (11C).Antibody-dependent-cellular-phagocytosis (ADCP) activity (left panel) ofserum from HSV-2 ΔgD-2 or control vaccinated mice 7 days post-boost wasquantified using THP-1 monocytic cell line and beads coated with HSV-2viral cellular lysates (v) or cellular lysates (c). IFN-γ levels (rightpanel) were measured in the supernatants 8 hr post THP-1 and Ab/beadincubation (11D). The % ADCP is calculated as percent of cells positivefor beads multiplied by the MFI of positive cells divided by 10⁶ (leftpanel). (*p<0.05; **p<0.01; ***p<0.001, HSV-2 ΔgD-2 vs.control-vaccinated group, student's t-test)

FIG. 12A-12H. Adaptive and innate immune cells are recruited to infectedskin by day 5 post-challenge in HSV-2 ΔgD-2 vaccinated mice. Skinsections from mice immunized with ΔgD-2 or VD60 lysates (control) andthen challenged with LD90 of SD90 or Bx³1.1 or unvaccinatedmock-infected controls were stained for CD3⁺ (T cells) (12A), B220⁺ (Bcells) (12B) or Iba1⁺ (pan macrophage) (12C); representativeimmunohistochemistry images following challenge with HSV-1(B³×1.1) orHSV-2(SD90) are shown. The percentage of CD3⁺ (12D), B220⁺ (12E), andIba1⁺ (12F) cells were enumerated by counting 3 random fields per mouse(5 mice per group). Skin sections were also stained for CD4⁺ (12G) andCD8⁺ (12H) by immunofluorescence and the percentage positive cellsquantified. Each symbol is the average of the 2 fields for individualmouse and the line represents mean; the dashed line represents countsfrom unvaccinated, mock-infected mice (3 fields averaging for 1 mouse)(*p<0.05, ΔgD-2-vs. control-vaccinated group by student's t-test).

FIG. 13. Control but not ΔgD-2 vaccinated mice have persistentneutrophil infiltration in skin biopsies. Skin sections of unimmunizedmock-infected mice or mice immunized with HSV-2 ΔgD-2 or VD60 lysates(control) and infected with HSV-1(B³×1.1) virus were harvested on Day 5post-challenge and stained for neutrophils using Ly6G (red). Nuclei arestained blue with DAPI.

FIG. 14A-14F. Mice immunized with ΔgD-2 have decreased inflammatorycytokines and chemokines in the skin compared to control immunized miceby day 5 post-challenge. Biopsies of skin from mice immunized with ΔgD-2or VD60 lysates (Control) at day 2 or day 5 post-challenge (orunimmunized, uninfected controls) were homogenized and evaluated for TNF(14A), IL-1β (14B), IL-6 (14C), CXCL9 (14D), CXCL10 (IP-10) (14E), andIL-33 (14F) (n=6 animals/group, line represents mean, dashed linerepresents counts from unimmunized, mock infected animals). (*p<0.05;**p<0.01; ***p<0.001, HSV-2 ΔgD-2-vaccinated group vs.control-vaccinated group students t-test.

DETAILED DESCRIPTION OF THE INVENTION

An isolated, recombinant herpes simplex virus-2 (HSV-2) is providedhaving a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof.

In an embodiment, the HSV-2 glycoprotein D comprises the amino acidsequence set forth in SEQ ID NO:1:MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGSTLAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (HSV-2 referencestrain HG52)

In an embodiment, the isolated, recombinant HSV-2 further comprises aherpes simplex virus-1 (HSV-1) glycoprotein D on a lipid bilayerthereof.

In an embodiment, the HSV-1 glycoprotein D comprises the amino acidsequence set forth in SEQ ID NO:2:MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRFRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMRRRTQKAPKRIRLPHIREDDQPSSHQPLFY (HSV-1 referencestrain F)

In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2U_(S6) gene. (For example, see Dolan et al. J Virol. 1998 March; 72(3):2010-2021. (PMCID: PMC109494) “The Genome Sequence of Herpes SimplexVirus Type 2” for HSV-2 genome and U_(S6) gene, hereby incorporated byreference in its entirety).

In an embodiment, the HSV-2 in which the HSV-2 glycoprotein D-encodinggene is deleted is an HSV-2 having a genome (prior to the deletion) asset forth in one of the following Genbank listed sequences: HSV-2(G)(KU310668), HSV-2(4674) (KU310667), B3×1.1 (KU310657), B3×1.2(KU310658), B3×1.3 (KU310659), B3×1.4 (KU310660), B3×1.5 (KU310661),B3×2.1 (KU310662), B3×2.2 (KU310663), B3×2.3 (KU310664), B3×2.4(KU310665), B3×2.5 (KU310666).

Also provided is a virion of an isolated, recombinant HSV-2 having adeletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof.

In an embodiment, the virion further comprises an HSV-1 or HSV-2glycoprotein D on a lipid bilayer thereof. In an embodiment, the HSV-2glycoprotein D-encoding gene is an HSV-2 U_(S6) gene. In an embodiment,the virion further comprises an HSV-1 glycoprotein D on a lipid bilayerthereof.

In an embodiment, the virus further comprises an HSV-1 or HSV-2glycoprotein D on a lipid bilayer thereof. In an embodiment, the HSV-2glycoprotein D-encoding gene is an HSV-2 U_(S6) gene. In an embodiment,the virus further comprises an HSV-1 glycoprotein D on a lipid bilayerthereof.

An isolated cell is provided comprising therein a recombinant HSV-2genome which does not comprise an HSV-2 U_(S6) gene.

In an embodiment, the recombinant HSV-2 genome is recombinant by virtueof having had a HSV-2 glycoprotein D gene deleted therefrom.

In an embodiment, the cell is a complementing cell which providesexpressed HSV 1 or 2 glycoprotein not encoded for by the recombinantHSV-2 genome. In an embodiment, the complementing cell comprises aheterologous nucleic acid encoding a HSV-1 or HSV-2 glycoprotein D. Inan embodiment, the cell expresses HSV-1 glycoprotein D on a membranethereof. In an embodiment of the cell, the HSV-1 glycoprotein D isencoded by the heterologous nucleic acid, which heterologous nucleicacid is a HSV-1 or HSV-2 glycoprotein D gene, or is a nucleic acidhaving a sequence identical to a HSV-1 or HSV-2 glycoprotein D gene.

Also provided is a vaccine composition comprising the recombinant HSV-2virus as described herein, or the virion as described herein. In anembodiment, the vaccine comprises an immunological adjuvant. In anembodiment, the vaccine does not comprise an immunological adjuvant. Inan embodiment of the vaccine, compositions or pharmaceuticalcompositions described herein comprising a recombinant HSV-2, the HSV-2is live.

Also provided is a composition comprising the recombinant HSV-2 virus asdescribed herein, or the virion as described herein, wherein the genomeof the virus or virion comprises at least a deletion of a second gene,wherein the second gene is necessary for HSV-2 viral replication orvirulence.

A pharmaceutical composition comprising the recombinant HSV-2 virus asdescribed herein, or the virion as described herein, and apharmaceutically acceptable carrier.

In an embodiment, the composition or pharmaceutical composition orvaccine is formulated so that it is suitable for subcutaneousadministration to a human subject. In an embodiment, the composition orpharmaceutical composition or vaccine is formulated so that it issuitable for intravaginal administration to a human subject. In anembodiment, the composition or pharmaceutical composition or vaccine isformulated so that it is suitable for intra-muscular, intra-nasal, ormucosal administration to a human subject.

Also provided is a method of eliciting an immune response in a subjectcomprising administering to the subject an amount of (i) the recombinantHSV-2 virus as described herein; (ii) a virion thereof as describedherein, (iii) the vaccine as described herein; (iv) a composition asdescribed herein; or (v) a pharmaceutical composition as describedherein, in an amount effective to elicit an immune response in asubject.

Also provided is a method of treating an HSV-2 infection in a subject ortreating a disease caused by an HSV-1, HSV-2 or co-infection in asubject comprising administering to the subject an amount of (i) therecombinant HSV-2 virus as described herein; (ii) a virion thereof asdescribed herein, (iii) the vaccine as described herein; (iv) acomposition as described herein; or (v) a pharmaceutical composition asdescribed herein, in an amount effective to treat an HSV-1, HSV-2 orco-infection or treat a disease caused by an HSV-1, HSV-2 orco-infection in a subject. In an embodiment, the methods comprisetreating an HSV-1 or HSV-2 pathology caused by an HSV-1, HSV-2 orco-infection. In an embodiment of the methods, the disease caused by anHSV-1, HSV-2 or co-infection is a genital ulcer. In an embodiment of themethods, the disease caused by an HSV-1, HSV-2 or co-infection isherpes, oral herpes, herpes whitlow, genital herpes, eczema herpeticum,herpes gladiatorum, HSV keratitis, HSV retinitis, HSV encephalitis orHSV meningitis.

In an embodiment of the methods herein regarding treating, orvaccinating for, an HSV-1, HSV-2 or co-infection (i.e. infection withboth HSV-1 and HSV-2), separate, individual, embodiments of treating anHSV-1 infection, treating an HSV-2 infection, treating a co-infection,vaccinating against an HSV-1 infection, vaccinating against an HSV-2infection, and vaccinating against a co-infection, are each provided.

Also provided is a method of vaccinating a subject for HSV-1, HSV-2 orco-infection comprising administering to the subject an amount of (i)the recombinant HSV-2 virus as described herein; (ii) a virion thereofas described herein, (iii) the vaccine as described herein; (iv) acomposition as described herein; or (v) a pharmaceutical composition asdescribed herein, in an amount effective to vaccinate a subject forHSV-1, HSV-2 or co-infection.

Also provided is a method of immunizing a subject against HSV-1, HSV-2or co-infection comprising administering to the subject an amount of (i)the recombinant HSV-2 virus as described herein; (ii) a virion thereofas described herein, (iii) the vaccine as described herein; (iv) acomposition as described herein; or (v) a pharmaceutical composition asdescribed herein, in an amount effective to immunize a subject againstHSV-1, HSV-2 or co-infection.

In an embodiment of the methods, the subject is administered asubcutaneous or intravaginal priming dose and is administered a seconddose subcutaneously or intravaginally. In an embodiment of the methods,the subject is administered as many subcutaneous or intravaginal primingdoses to elicit anti-HSV antibodies and T cells.

Also provided is a method of producing a virion of a recombinant herpessimplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoproteinD-encoding gene in the genome thereof and comprising an HSV-1 or HSV-2glycoprotein D on a lipid bilayer thereof, comprising infecting a cellcomprising a heterologous nucleic acid encoding a HSV-1 or HSV-2glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) havinga deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof under conditions permitting replication of the recombinantherpes simplex virus-2 (HSV-2) and recovering a HSV-2 virion produced bythe cell.

In an embodiment, the cell expresses HSV-1 or HSV-2 glycoprotein D on amembrane thereof.

Also provided is a recombinant nucleic acid having the same sequence asa genome of a wild-type HSV-2 except that the recombinant nucleic aciddoes not comprise a sequence encoding an HSV-2 glycoprotein D. In anembodiment, the recombinant nucleic acid is a DNA. In an embodiment, therecombinant nucleic acid is an RNA.

Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2)having a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof for treating or preventing an HSV-1, HSV-2 or co-infection in asubject. In an embodiment, the isolated, recombinant HSV-2 furthercomprises a herpes simplex virus-1 (HSV-1) or herpes simplex virus-2(HSV-2) glycoprotein D on a lipid bilayer thereof. In an embodiment ofthe isolated, recombinant HSV-2, the HSV-2 glycoprotein D-encoding geneis an HSV-2 U_(S6) gene.

Also provided is a virion of an isolated, recombinant HSV-2 having adeletion of an HSV-2 glycoprotein D-encoding gene in the genome thereoffor treating or preventing an HSV-1, HSV-2 or co-infection in a subject.In an embodiment, the virion further comprises an HSV-1 or HSV-2glycoprotein D on a lipid bilayer thereof. In an embodiment, the HSV-2glycoprotein D-encoding gene is an HSV-2 U_(S6) gene.

In an embodiment, of the virus or virion as described, the HSV-1, HSV-2or co-infection causes a genital ulcer.

An isolated, recombinant herpes simplex virus-2 (HSV-2) is providedhaving a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof.

In an embodiment, the isolated, recombinant HSV-2 further comprises asurface glycoprotein on a lipid bilayer thereof which is a herpessimplex virus-1 (HSV-1) glycoprotein D. In an embodiment, the isolated,recombinant HSV-2 further comprises a non-HSV-2 viral surfaceglycoprotein on a lipid bilayer thereof. In an embodiment, the isolated,recombinant HSV-2 further comprises a bacterial surface glycoprotein ona lipid bilayer thereof. In an embodiment, the isolated, recombinantHSV-2 further comprises a parasitic surface glycoprotein on a lipidbilayer thereof, wherein the parasite is a parasite of a mammal.

In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 US6gene. In an embodiment, the surface glycoprotein is encoded by atransgene that has been inserted into the genome of the recombinantHSV-2. In an embodiment, the surface glycoprotein is present on a lipidbilayer thereof by way of infecting a cell with a recombinant HSV-2having a deletion of an HSV-2 glycoprotein D-encoding gene, wherein thecell is or has been transfected to express the surface glycoprotein on acell membrane thereof, and wherein the recombinant HSV-2 comprising thesurface glycoprotein present on a lipid bilayer is produced from thecell. In an embodiment, the viral glycoprotein is from a HIV, anenterovirus, a RSV, an influenza virus, a parainfluenza virus, Pigcorona respiratory virus, a rabies virus, a Lassa virus, a bunyavirus, aCMV, or a filovirus. In an embodiment, the glycoprotein is an HIV gp120.In an embodiment, the filovirus is an ebola virus. In an embodiment, thevirus is HIV, a M. tuberculosis, a chlamydia, Mycobacterium ulcerans, M.marinum, M. leprae, M. absenscens, Neisseria gonnorhea, or a Treponeme.In an embodiment, the Treponeme is Treponeme palidum.

Also provided is a virion of an isolated, recombinant HSV-2 having adeletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof.

In an embodiment, the virion of the isolated, recombinant HSV-2 furthercomprises a surface glycoprotein on a lipid bilayer thereof which is aherpes simplex virus-1 (HSV-1) glycoprotein D. In an embodiment, thevirion of the isolated, recombinant HSV-2 further comprises a non-HSV-2viral surface glycoprotein on a lipid bilayer thereof. In an embodiment,the virion of the isolated, recombinant HSV-2 further comprises abacterial surface glycoprotein on a lipid bilayer thereof. In anembodiment, the virion of the isolated, recombinant HSV-2 furthercomprises a parasitic surface glycoprotein on a lipid bilayer thereof,wherein the parasite is a parasite of a mammal. In an embodiment, theHSV-2 glycoprotein D-encoding gene is an HSV-2 U_(S6) gene. In anembodiment, the surface glycoprotein is encoded by a transgene that hasbeen inserted into the genome of the recombinant HSV-2 of the virion. Inan embodiment, the surface glycoprotein is present on a lipid bilayerthereof by way of infecting a cell with a recombinant HSV-2 having adeletion of an HSV-2 glycoprotein D-encoding gene, wherein the cell isor has been transfected to express the surface glycoprotein on a cellmembrane thereof, and wherein the recombinant HSV-2 comprising thesurface glycoprotein present on a lipid bilayer is produced from thecell. In an embodiment, the virion has been recovered from such. In anembodiment, the viral glycoprotein is from a HIV, an enterovirus, a RSV,an influenza virus, a parainfluenza virus, Pig corona respiratory virus,a rabies virus, a Lassa virus, a bunyavirus, a CMV, or a filovirus. Inan embodiment, the glycoprotein is an HIV gp120. In an embodiment, thefilovirus is an ebola virus. In an embodiment, the virus is HIV, a M.tuberculosis, a chlamydia, Mycobacterium ulcerans, M. marinum, M.leprae, M. absenscens, Neisseria gonnorhea, or a Treponeme. In anembodiment, the Treponeme is Treponeme palidum.

Also provided is an isolated cell comprising therein a virus asdescribed herein or a virion as described herein, wherein the cell isnot present in a human being. In an embodiment of the cell, the cellcomprises a heterologous nucleic acid encoding a HSV-1 glycoprotein D.In an embodiment of the cell, the cell expresses HSV-1 glycoprotein D ona membrane thereof.

In an embodiment of the cell, the HSV-1 glycoprotein D is encoded by theheterologous nucleic acid, which heterologous nucleic acid is a HSV-1glycoprotein D gene, or is a nucleic acid having a sequence identical toa HSV-1 glycoprotein D gene.

A vaccine composition comprising a virus as described herein, or avirion as described herein. In an embodiment of the vaccine composition,the vaccine composition comprises an immunological adjuvant.

Also provided is a composition comprising a virus as described herein,or a virion as described herein, wherein the genome of the virus orvirion comprises at least a deletion of a second gene, wherein thesecond gene is necessary for HSV-2 viral replication. In an embodiment,the composition comprises serum from, or is derived from serum from, amammal into which the virus or virion has been previously introduced soas to elicit an immune response.

Also provided is pharmaceutical composition comprising a virus asdescribed herein, or a virion as described herein, and apharmaceutically acceptable carrier.

Also provided is a method of eliciting an immune response in a subjectcomprising administering to the subject an amount of (i) a virus asdescribed herein; (ii) a virion as described herein, (iii) a vaccine asdescribed herein; (iv) a composition as described herein; or (v) apharmaceutical composition as described herein, in an amount effectiveto elicit an immune response in a subject.

Also provided is a method of treating an HSV-2 infection in a subject ortreating a disease caused by an HSV-2 infection in a subject comprisingadministering to the subject an amount of (i) a virus as describedherein; (ii) a virion as described herein, (iii) a vaccine as describedherein; (iv) a composition as described herein; or (v) a pharmaceuticalcomposition as described herein, in an amount effective to treat anHSV-2 infection or treat a disease caused by an HSV-2 infection in asubject.

Also provided is a method of vaccinating a subject for HSV-2 infectioncomprising administering to the subject an amount of (i) a virus asdescribed herein; (ii) a virion as described herein, (iii) a vaccine asdescribed herein; (iv) a composition as described herein; or (v) apharmaceutical composition as described herein, in an amount effectiveto vaccinate a subject for HSV-2.

Also provided is a method of immunizing a subject against HSV-2infection comprising administering to the subject an amount of (i) avirus as described herein; (ii) a virion as described herein, (iii) avaccine as described herein; (iv) a composition as described herein; or(v) a pharmaceutical composition as described herein, in an amounteffective to immunize a subject against HSV-2.

HSV-2 and HSV-1 diseases are known in the art, and are also describedherein. Both treatment and prevention of HSV-2 and HSV-1 diseases areeach separately encompassed. Also treatment or prevention of a HSV-2 andHSV-1 co-infection are covered. Prevention is understood to meanamelioration of the extent of development of the relevant disease orinfection in a subject treated with the virus, virion, vaccine orcompositions described herein, as compared to an untreated subject.

Also provided is a method of producing a virion of a recombinant herpessimplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoproteinD-encoding gene in the genome thereof and comprising an HSV-1glycoprotein D on a lipid bilayer thereof, comprising infecting a cellcomprising a heterologous nucleic acid encoding a HSV-1 glycoprotein Dwith a recombinant herpes simplex virus-2 (HSV-2) having a deletion ofan HSV-2 glycoprotein D-encoding gene in the genome thereof underconditions permitting replication of the recombinant herpes simplexvirus-2 (HSV-2) and recovering a recombinant HSV-2 virion comprising anHSV-1 glycoprotein D on a lipid bilayer thereof produced by the cell.

Also provided is a method of producing a virion of a recombinant herpessimplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoproteinD-encoding gene in the genome thereof and comprising a non-HSV-2 surfaceglycoprotein on a lipid bilayer thereof, comprising infecting a cellcomprising a heterologous nucleic acid encoding the non-HSV-2 surfaceglycoprotein with a recombinant herpes simplex virus-2 (HSV-2) having adeletion of an HSV-2 glycoprotein D-encoding gene in the genome thereofunder conditions permitting replication of the recombinant herpessimplex virus-2 (HSV-2) and recovering a recombinant HSV-2 virioncomprising a non-HSV-2 surface glycoprotein on a lipid bilayer thereofproduced by the cell.

Also provided is a recombinant nucleic acid is provided having the samesequence as a genome of a HSV-2 except that the sequence does notcomprise a sequence encoding an HSV-2 glycoprotein D.

Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2)having a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof for treating or preventing an HSV-2 infection in a subject.

Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2)having a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof for treating or preventing an HSV-1 infection in a subject.

Also provided is a virion of an isolated, recombinant HSV-2 having adeletion of an HSV-2 glycoprotein D-encoding gene in the genome thereoffor treating or preventing an HSV-2 infection in a subject.

Also provided is a method of treating an HSV-1 infection, or HSV-1 andHSV-2 co-infection, in a subject, or treating a disease caused by anHSV-2 infection or HSV-1 and HSV-2 co-infection in a subject comprisingadministering to the subject an amount of (i) a virus as describedherein; (ii) a virion as described herein, (iii) a vaccine as describedherein; (iv) a composition as described herein; or (v) a pharmaceuticalcomposition as described herein, in an amount effective to treat anHSV-2 infection or treat a disease caused by an HSV-2 infection in asubject or an amount effective to treat an HSV-1 and HSV-2 co-infectionor treat a disease caused by an HSV-1 and HSV-2 co-infection in asubject.

Also provided is a method of vaccinating a subject for an HSV-1infection, or HSV-1 and HSV-2 co-infection, comprising administering tothe subject an amount of (i) a virus as described herein; (ii) a virionas described herein, (iii) a vaccine as described herein; (iv) acomposition as described herein; or (v) a pharmaceutical composition asdescribed herein, in an amount effective to vaccinate a subject for anHSV-1 infection, or HSV-1 and HSV-2 co-infection.

Also provided is a method of immunizing a subject against an HSV-1infection, or HSV-1 and HSV-2 co-infection, comprising administering tothe subject an amount of (i) a virus as described herein; (ii) a virionas described herein, (iii) a vaccine as described herein; (iv) acomposition as described herein; or (v) a pharmaceutical composition asdescribed herein, in an amount effective to immunize a subject againstan HSV-1 infection, or HSV-1 and HSV-2 co-infection.

In an embodiment of the methods herein for immunizing, vaccinating oreliciting an immune response, passive transfer of the virion or virus orthe antibodies or immune factors induced thereby may be effected fromone subject to another. The relevant product may be treated afterobtention from one subject before administration to a second subject. Ina preferred embodiment of the inventions described herein, the subjectis a mammalian subject. In an embodiment, the mammalian subject is ahuman subject.

Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2)having a deletion of an HSV-2 glycoprotein D-encoding gene in the genomethereof and further comprising a heterogenous antigen of a pathogen. Inan embodiment, the heterogenous antigen is a protein, peptide,polypeptide or glycoprotein. In an embodiment, the heterogenous antigenheterogenous antigen with respect to HSV-2, but is an antigen found onor in the relevant “pathogen.” Pathogens, viral and bacterial, aredescribed herein. In an embodiment, the pathogen is a bacterial pathogenof a mammal or a viral pathogen of a mammal. In an embodiment, theantigen or the transgene encoding the pathogen is not actually taken orphysically removed from the pathogen, but nevertheless has the samesequence as the pathogen antigen or encoding nucleic acid sequence. Inan embodiment, the isolated, recombinant HSV-2 comprises a heterogenousantigen of a pathogen on a lipid bilayer thereof. In an embodiment ofthe isolated, recombinant HSV-2, the pathogen is bacterial or viral. Inan embodiment, the pathogen is a parasite of a mammal. In an embodiment,the HSV-2 glycoprotein D-encoding gene is an HSV-2 U_(S6) gene. In anembodiment, the isolated, recombinant HSV-2, the heterogenous antigen isencoded by a transgene that has been inserted into the genome of therecombinant HSV-2.

Also provided is a method of inducing antibody dependent cell mediatedcytotoxicity (ADCC) against an antigenic target in a subject comprisingadministering to the subject an isolated, recombinant herpes simplexvirus-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encodinggene in the genome thereof and further comprising a heterogenous antigenon a lipid bilayer thereof in an amount effective to induce antibodydependent cell mediated cytotoxicity (ADCC) against an antigenic target.

Recombinant HSV-2 ΔgD^(−/+ gD−/+) expressing the appropriate transgeneswill selectively induce antibodies and cellular immune responses thatprotect against skin or mucosal infections by pathogens.

In an embodiment, the heterogenous antigen is a surface antigen.

In an embodiment, the transgene encodes an antigen from an HIV, a M.tuberculosis, a chlamydia, Mycobacterium ulcerans, M. marinum, M.leprae, M. absenscens, Neisseria gonnorhea, or a Treponeme. In anembodiment, the Treponeme is Treponeme palidum. In an embodiment, thetransgene is a M. tuberculosis biofilm-encoding gene. In an embodiment,the transgene is an HIV gp120-encoding gene.

In an embodiment, the heterogenous antigen is a surface antigen of theantigenic target. In an embodiment, the heterogenous antigen is aparasite antigen. In an embodiment, the heterogenous antigen is abacterial antigen or a viral antigen.

In an embodiment, the antigenic target is a virus and is a Lassa virus,a human immunodeficiency virus, an RSV, an enterovirus, an influenzavirus, a parainfluenza virus, pig corona respiratory virus, alyssavirus, a bunyavirus, or a filovirus.

In an embodiment, the antigenic target is a bacteria and is Mycobateriumtuberculosis, M. ulcerans, M. marinum, M. leprae, M. absenscens,Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum.

In an embodiment, the isolated, recombinant HSV-2 transgene is a M.tuberculosis biofilm-encoding gene or wherein the transgene is an HIVgp120-encoding gene.

In a preferred embodiment of the methods described herein, the subjectis a human. In an embodiment of the methods described herein, thesubject has not yet been infected with HSV-1, HSV-2 or co-infection. Inan embodiment of the methods described herein, the subject has beeninfected with HSV-1, HSV-2 or co-infection.

As described herein, a co-infection means a co-infection with HSV-1 andHSV-2.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS Example 1

Herein a genetically engineered deletion mutant of the gD (U_(S6)) geneof HSV-2 is disclosed and its safety, immunogenicity, and vaccineefficacy evaluated against intravaginal HSV-2 challenge in the mouseinfection model. The gD gene was replaced with a DNA fragment encodingthe green fluorescent protein (gfp) and Vero cells expressing HSV-1 gD(VD60 cells) were transfected with this construct and screened forhomologous recombinant virus that formed green plaques. Molecularanalysis revealed that a precise recombination had been engineered,which replicates in the complementing VD60 cells to high titers but isnoninfectious when propagated on non-complementing cells. Intravaginalchallenge of wild-type or SCID mice with 10⁷ pfu/mouse of thecomplemented gD-null virus (designated herein as HSV-2 ΔgD^(−/+) for thevirus that is genotypically gD deleted, but phenotypically complementedby growth on VD60 cells) revealed no virulence, whereas doses as low as10⁴ pfu/mouse of parental wild-type virus were 100% lethal. Moreoverimmunization of mice with HSV-2 ΔgD^(−/+) yielded complete protectionagainst intravaginal challenge with a clinical isolate of HSV-2. Robusthumoral and cellular immunity elicited by HSV-2 ΔgD^(−/+) was measuredand it is concluded that gD is required for productive infection in vivoand that an attenuated strain deleted in this essential glycoproteinelicits protective immunity against HSV-2. Thus, HSV-2 ΔgD^(−/+) is apromising vaccine for prevention or treatment of genital herpes.

Mechanisms and correlates of protection elicited by HSV-2 ΔgD−/+. A gD-2null virus was generated, and it was demonstrated that it is highlyattenuated in both immunocompetent and immunocompromised mice and whentested as a vaccine candidate, induced a protective immune responseagainst intravaginal challenge with HSV-2. Subcutaneous immunizationswith HSV-2 ΔgD−/+ will induce humoral and cellular immune responses thatare required for protection against intravaginal challenge with bothserotypes of HSV (HSV-2 and HSV-1).

HSV-2 ΔgD−/+ initiates an abortive infection: An HSV-2 strain that isdeleted for U_(S)6 was constructed to assess its contribution in earlysignaling events occurring during cell infection [41]. This virus isincapable of infecting host cells, unless it is grown on agD-complementing cell line (e.g. VD60 cells encoding gD-1 [40, 41]) thatencodes U_(S)6 under the control of its endogenous promoter (forexample, in an embodiment, the gD-1 promoter). Indeed, HSV-2 ΔgDparticles isolated from non-complementing cells do not infect epithelial(FIG. 1) or neuronal cells (SK-N-SH, not shown). However, if propagatedin VD60 cells a phenotypically complemented virus (ΔgD−/+) is obtained,which is fully capable of infecting cells that are common targets forwild-type HSV-2. However, after infection with ΔgD−/+no infectiousparticles or viral plaques (pfu) are produced from these cells and thevirus fails to spread from infected to uninfected cells, reflecting therequirement for gD in these processes; thus it is an abortive infection.

HSV-2 ΔgD−/+ is safe in the murine infection model: ΔgD−/+ was evaluatedfor safety in vivo in wild-type and severe combined immunodeficiency(SCID) mice by inoculating high doses subcutaneously or intravaginally.Mice inoculated intravaginally with 10⁷ pfu of ΔgD−/+(titered oncomplementing cells) did not manifest any signs of virus-inducedpathology throughout the experiments, whereas animals inoculated with1,000-fold less wild-type virus (10⁴ pfu) succumbed to HSV-2 disease anddied starting Day 8 after inoculation (FIG. 2A). Mice inoculatedintravaginally with 10⁷ pfu of ΔgD−/+ did not manifest any signs ofvirus-induced epithelial or neurological disease throughout theexperiments (FIGS. 2B and 2C). No infectious virus was recovered fromgenital tract tissue or DRGs, as determined by plaque assay orco-cultivation of DRGs with Vero cells (not shown).

HSV-2 ΔgD−/+ elicits systemic and mucosal antibodies to HSV-2: Miceinoculated and boosted subcutaneously (sc.-sc.) with ΔgD−/+ orinoculated subcutaneously and boosted intravaginally (sc.-i.vag.) withthis candidate vaccine strain (10⁶ pfu/mouse) elicited a humoral immuneresponse to HSV-2 as evidenced by an increase in serum and vaginalwashes anti-HSV-2 antibodies (FIGS. 3A and 3B). The control animals wereimmunized with an uninfected VD60 cell lysate (referred to as Control).The antibodies were measured by ELISA using infected cell lysates as theantigen (response to uninfected cell lysates subtracted as background).Noteworthy, the magnitude of the antibody response differs depending onthe route of immunization. Indeed, s.c.-s.c. immunization elicitedsignificantly more serum and vaginal wash antibodies to HSV-2 thans.c.-i.vag. immunization. This finding suggests that the vaginal washantibodies likely represent transudate of IgG from the blood and suggestthat sc.-sc. is a more appropriate route for eliciting high levels ofsystemic and local IgG antibodies to HSV-2. Additionally, Miceinoculated and boosted subcutaneously (sc.-sc.) with ΔgD−/+ (10⁶pfu/mouse) elicited a neutralizing anti-HSV-2 as evidenced by in vitroneutralization of Vero cell monolayers with virus and sera from thesemice (FIG. 3C).

HSV-2 ΔgD−/+ elicits HSV-2-specific T cell activation:gB498-505-specific transgenic CD8+ T cells (gBT-I) were transferred intoC57BL/6 mice prior to vaccination. Vaccinated mice were inoculated with10⁶ pfu ΔgD−/+ or with VD60 cell lysates (Control). Spleens wereharvested on Day 14 after the boost and quantified by flow cytometryusing counting beads (CountBright™, Lifetechnologies) (FIG. 4A). At thesame day, spleens were stained for memory surface markers and analyzedby flow cytometry (FIG. 4B). Finally, splenocytes harvested the same daywere re-stimulated in vitro for 6 hours with the agonistgB498-505-peptide and intracellular cytokine staining was performed tomeasure IFN-γ production by these cells. Immunization with ΔgD−/+increased the IFN-γ production in the vaccinated compared to controlmice (FIG. 4C). The response in control mice presumably reflects thepersistence of the gBT-I T cells in naïve mice after transfer. Similarresults were obtained using multiplex cytokine analyses for supernatantsof splenocytes re-stimulated in vitro with gB498-505-peptide (notshown). These findings demonstrate that the vaccine induces T cellresponses.

Mice immunized with HSV-2 ΔgD−/+ are protected against intravaginalHSV-2 lethal challenge: Animals vaccinated with HSV-2 ΔgD−/+ eithersc.-sc. or sc.-i.vag. suffer less body weight after intravaginal lethaldose challenges equivalent to LD₉₀ (5×10⁴ pfu/mouse) and survivechallenges, whereas mice immunized with the VD60 control lysatesuccumbed to disease by Day 10 (FIGS. 5A and 5B). The vaccines alsoprovided complete protection against 10 times the LD₉₀ (5×10⁵ pfu/mouse,data not shown). This protection was associated with significantlyreduced epithelial disease scores (FIG. 5C) and the complete absence ofneurological signs (FIG. 5D). Scoring was performed as previouslydescribed [44]. Furthermore, significantly less virus was recovered invaginal washes in ΔgD−/+-immunized mice, as compared to control mice atday 2 post-vaginal challenge suggesting rapid clearance (FIG. 5E).Moreover no infectious virus was recovered in Day 4 vaginal washes (FIG.5E) or in vaginal tissue or DRGs isolated on Day 5 after challenge (FIG.5F). The latter suggest that the vaccine prevents virus from reachingand/or replicating in the DRG.

Immunization with HSV-2 ΔgD−/+ prevents inflammation at the infectionsite after challenge with virulent HSV-2: Mice vaccinated with HSV-2ΔgD−/+ and intravaginally challenged with virulent HSV-2 displaysignificantly less inflammatory cytokines at the infection site ascompared to animals inoculated with VD60 lysates (Control). Indeed,vaccinated mice secreted significantly less TNF-α (FIG. 6A), IL-6 (FIG.6B) and IL-1β (FIG. 6C) in vaginal washes at Day 2 and 7 post-infectionthan Control mice. Noteworthy, increased levels of inflammatorycytokines are associated with increased HIV replication and shedding atthe genitalia in the co-infected with HSV-2 and HIV [45, 46]. A similarphenomenon is also observed in vitro [47].

Immunization with HSV-2 ΔgD−/+ recruits T cells to the infection siteand associated LNs. Mice immunized sc.-sc. with ΔgD−/+ displayedincreased percentages of activated anti-HSV-2 gBT-I CD8+(FIG. 7A) andCD4+ T cells (FIG. 7B) in sacral lymph nodes (LNs) after challenge withvirulent HSV-2. Mice immunized sc.-i.vag. with ΔgD−/+ displayedincreased numbers of anti-HSV-2 gBT-I CD8+(FIG. 7C) and CD4+ T cells(FIG. 7D) in the vagina after challenge with virulent HSV-2 suggestingthat vaccination with ΔgD−/+ recruits anti-HSV-2 CD8+ T cells andactivated CD4+ T cells (likely anti-HSV-2) to the infection site andassociated lymph nodes.

In further experiments, immunization with HSV-2-ΔgD^(−/+gD-1) was foundto confer protection in C57BL/6 and Balb/C to vaginal challenge withvirulent HSV-2. In addition, intravaginal HSV-2 challenged ΔgD^(−/+gD-1)immunized mice had no detectable HSV-2 in vaginal or neural tissue at 5days post-challenge. HSV-2 ΔgD−/+gD-1 sc.sc. antibodies were found torecognize numerous HSV-2 proteins (both gD and gB) unlike HSV-2morbid-bound mice. Serum antibodies from vaccinated animals showedneutralization of HSV-1 and HSV-2 in vitro. Moreover, serum fromΔgD−/+gD-1 vaccinated mice elicited Antibody Dependent CellularCytotoxicity (ADCC) of HSV-2 infected cells in vitro.

In summary, HSV-2 ΔgD−/+gD-1 is attenuated and completely safe in wt andSCID mice. Recombinant HSV-2 ΔgD−/+gD-1 protected against lethal HSV-2intravaginal and HSV-2/HSV-1 skin infection. Protection was observed intwo different mouse strains. There was no detectable infection, andsterilizing immunity. Also observed was induction of HSV-2 specific CD8+T cells and systemic and mucosal HSV Abs. IgG2a and IgG2b were thepredominant anti-HSV isotype. Also observed was FcyRIII/II-dependentADCC. Surprisingly, passive transfer of immune serum protects naïvemice, and FcRn and FcyR knockout mice were not protected with immunesera.

Discussion

The World Health Organization estimated that over 500 million peoplewere infected with herpes simplex virus type 2 (HSV-2) worldwide withapproximately 20 million new cases annually [1]. Infection riskincreases with age and because the virus establishes latency withfrequent subclinical or clinical reactivation, the impact of infectionis lifelong. Alarmingly, HSV-2 significantly increases the risk ofacquiring and transmitting HIV [2-4]. The prevalence of HSV-2 variesamong global regions, fluctuating from 8.4% for Japan up to 70% forsub-Saharan Africa, a region where HIV prevalence is epidemic [5, 6]. Inthe US the prevalence of HSV-2 is ˜16% and that of HSV-1 has declined to˜54%. The decreasing prevalence of HSV-1 in the US (and other Europeannations) is linked to an increase in genital HSV-1 as evidenced byresults in the recent disappointing glycoprotein D (gD) subunit vaccinetrial in which the majority of cases of genital herpes disease werecaused by HSV-1 [7-9]. While HSV-1 is associated with fewer recurrencesand less genital tract viral shedding compared to HSV-2, both serotypesare transmitted perinataly and cause neonatal disease; neonatal diseaseis associated with high morbidity and mortality even with acyclovirtreatment [10-12]. The morbidity associated with genital herpes, itssynergy with the HIV epidemic, and its direct medical cost, whichsurpasses 500 million dollars in the US alone, highlight the imperativeto develop a safe and effective vaccine [13].

Subunit formulations consisting of viral envelope glycoproteins combinedwith adjuvants have predominated the HSV-2 vaccine field for nearly 20years and the majority of clinical trials have focused on this strategy[8, 14-19]. Although subunit preparations are safe and elicitneutralizing antibodies, these formulations provided little efficacyagainst HSV-2 infection or disease in clinical trials [8, 14].Surprisingly, an HSV-2 gD subunit vaccine provided protection againstgenital HSV-1, but not HSV-2 [8, 20]. Subsequent studies found thatserum HSV-2 gD antibody levels correlated with protection against HSV-1,suggesting that the antibody titers required for HSV-2 protection may behigher than those needed to protect against HSV-1 [21]. In contrast,cell mediated immunity (intracellular cytokine responses to overlappinggD peptides) did not correlate with protection against either serotype[21]. The vaccine elicited CD4⁺, but not CD8⁺ T cell responses, butthere were no differences in CD4⁺ T cell responses between vaccinatedinfected and uninfected women [21]. Genital tract or other mucosalantibody responses were not measured. An HSV-2 vaccine candidate with gHdeleted from the genome failed to reduce the frequency of viralrecurrences in a clinical trial conducted among seropositive subjects,although the vaccine was not evaluated for efficacy against primaryinfection [29].

Clinical studies showing increased rates of HSV-2 reactivation inHIV-infected patients combined with the failure of the gD subunitvaccine to elicit any CD8⁺ T cell response despite the induction ofneutralizing serum antibodies suggest that an effective vaccine mustalso elicit protective T cell responses [28, 30-32]. The importance of Tcells is further highlighted by studies showing selective retention ofHSV-1 reactive T-cells in human trigeminal ganglia. CD4⁺ and CD8⁺ Tcells were identified surrounding neurons and, while there washeterogenity in the viral proteins targeted, the tegument protein,virion protein 16 (VP16), was recognized by multiple trigeminal gangliaT cells in the context of diverse HLA-A and -B alleles; these findingssuggest that tegument proteins may be important immunogens [33].Similarly, cytotoxic T cells directed at tegument proteins were alsoidentified in studies of humans latently infected with HSV-2 [34]. CD8⁺T cells (including CD8αα⁺ T cells) persist in genital skin and mucosa atthe dermal-epidermal junction following HSV reactivation suggesting thatthey play a role in immune control [35].

Herein is disclosed an engineered an HSV-2 virus genetically deleted fornative HSV-2 gD. The HSV-2 gD gene encodes an envelope glycoproteinessential for viral entry and cell-to-cell spread. Glycoprotein D alsobinds to tumor necrosis factor receptor superfamily member 14(TNFRSF14), an immune-regulatory switch also known as herpesvirus entrymediator (HVEM). Because HVEM harbors docking sites for more than oneligand and signaling differs depending on whether these molecules bindto HVEM in cis or in trans, gD may have modulatory effects on immunecells [36, 37]. Indeed, recent studies suggest that gD competes with thenatural ligands for this receptor and modulates the cytokine response tothe virus [38, 39]. The gD gene was replaced with a DNA fragmentencoding the green fluorescent protein (gfp) and transformedcomplementing Vero cells expressing HSV-1 gD (VD60 cells [40]) (e.g.gD-1 under gD-1 promoter) with this construct were screened forhomologous recombinant virus that formed green plaques. The mutant virusreplicates in the complementing Vero cell line to high titers(designated HSV-2 when passaged on complementing cells), but isnoninfectious in non-complementing cells (designated HSV-2 ΔgD^(−/−)when isolated from non-complementing cells). This virus was purified andcharacterized in vitro [41]. Intravaginal or subcutaneous inoculation ofimmunocompetent or immunocompromised (SCID) mice revealed no virulencecompared to the lethal infection caused by parental wild-type virus.Immunization (subcutaneous prime followed by a single boost administeredeither subcutaneously or intravaginally) was 100% protective againstintravaginal challenge with virulent HSV-2. Robust humoral and cellularimmunity was elicited by HSV-2 ΔgD^(−/+) and it was concluded thatU_(S)6 (gD-2) is required for productive infection in vivo. This liveattenuated viral strain will provide sterilizing immunity against HSV.Also passive serum or serum product transfer can be employed.

Example 2

The ability of the vaccine to protect against clinical HSV-1 and HSV-2isolates was further confirmed, as was the local immune response at thesite of infection. Classically, HSV primarily infects genital or oralnucleated epidermal cells due to breaches in the skin or mucocutaneouslayers in humans (75). To more closely model human HSV infection, a skinscarification model was used for these studies, which displays viralkinetics and histopathology similar to humans (76).

Results

Immunization with low doses of ΔgD-2 are protective against lethalintravaginal and skin challenges. Previous studies were conducted with5×10⁶ PFU (titer determined on complementing VD60 cells) of ΔgD-2 as thevaccine inoculum. A dose de-escalation study was conducted to determineprotection at lower doses of vaccination. C57BL/6 mice (5 mice/group)were subcutaneously (sc) primed and boosted 21 days later with 5×10⁶PFU, 5×10⁵ PFU, or 5×10⁴ PFU of ΔgD-2. Three weeks later the mice werechallenged either intravaginally or by skin scarification with an LD₉₀(5×10⁵ PFU) of HSV-2(4674), a previously described clinical isolate(77). All HSV-2 ΔgD-2 immunized mice survived (5/5 per group), whereasall of the control-vaccinated mice (immunized with VD60 cell lysate)succumbed to disease (FIG. 8A, 8B). Although the mice vaccinated withthe lowest dose (5×10⁴) showed mild epithelial disease, no signs ofneurological disease were observed and all animals completely recoveredby day 14 in both the vaginal and skin challenge models. HSV antibodies(measured by ELISA) were detected in the serum of all vaccinated miceone week after boost, but not prime, and the antibody titer increasedwith administration of higher vaccine doses (FIG. 8C).

Mice immunized with ΔgD-2 are protected from high viral challenges ofvirulent HSV-1 and HSV-2 clinical isolates. To evaluate if the ΔgD-2vaccine protects against diverse HSV-1 and HSV-2 strains, five HSV-1were obtained (denoted B3×1.1-B3×1.5) and five HSV-2 (denotedB3×2.1-B3×2.5) clinical isolates from the Clinical Virology Lab atMontefiore located in the Bronx, N.Y. as well as a South African HSV-2clinical isolate (SD₉₀). The isolates were grown on Vero cells and werepassaged no more than three times before sequencing and phenotyping.Illumina sequencing showed that the strains exhibited substantialgenetic diversity with pairwise distances as high as 6.3% between B3×1.5and the other B3×1 isolates and 5.0% between B3×2.2 and the other B3×2isolates. In vivo virulence of each clinical strain was compared tolaboratory strains by challenging Balb/C mice using the skinscarification model with 1×10⁵ PFU of the HSV-1 strains or 5×10⁴ PFU ofHSV-2 strains. The clinical isolates demonstrated a range of virulencewith B3×1.1, B3×1.3, B3×2.3, and SD₉₀ inducing more rapid disease withthe highest morbidity in naïve mice. Similar results were observed inthe vaginal challenge model with the same 4 isolates exhibiting the mostvirulent disease (not shown). Interestingly, no differences between theisolates were observed by in vitro single and multistep growth curves onVero cells.

To assess if ΔgD-2 protected against the different isolates, C57BL/6 orBalb/C mice were primed and boosted with 5×10⁶ PFU/mouse of ΔgD-2 (orVD60 lysate as the control immunogen) and then challenged with an LD90dose of the 4 more virulent clinical isolates (Table 1) using the skinscarification model. All ΔgD-2 vaccinated mice survived challenge (n=7C57BL/6 mice per group; FIG. 3A, and n=5 Balb/C mice per group, FIG.9B). While some mice exhibited mild epithelial disease, which peaked onDay 4, the majority of animals had fully recovered by day 8post-challenge. No signs of neurological disease were detected in any ofthe mice at any time point.

TABLE 1 HSV strains used in vaccine efficacy studies Viral Strain Originof Isolate HSV Serotype Lethal Dose₉₀* B³x1.1 United States Type 1 5 ×10⁵ pfu B³x1.3 United States Type 1 1 × 10⁵ pfu SD90 South Africa Type 25 × 10⁴ pfu B³x2.3 United States Type 2 1 × 10⁵ pfu 4674 United StatesType 2 5 × 10⁵ pfu Note: *Plaque forming units that cause 90% morbidityin Balb/C mice skin challenge model

To further evaluate the robustness of the immune response, the challengedose was increased in the C57BL/6 mice to 10× and 100× the LD₉₀ doses ofSD90 and 10× the LD₉₀ of B3×1.1. All of the ΔgD-2 vaccinated micesurvived (FIG. 9D) with no signs of neurological disease. The ΔgD-2vaccinated mice had significantly reduced virus detected in skinbiopsies by day 5 post-challenge with the majority having no viralplaques detected (FIG. 10A, n=3 mice per group). Consistent with therapid clearance of virus, histopathology of skin biopsies revealedulceration and necrosis covering 75-95% of the epithelium incontrol-vaccinated mice compared to <10% epithelial necrosis andulceration in both the ΔgD-2 vaccinated mice and mock (unvaccinated,uninfected) treated mice. Moreover, there was no replicating or latentvirus detected by plaque assay (FIG. 10B) or qPCR (FIG. 10C),respectively, in DRGs isolated on Day 14 post-challenge in the ΔgD-2vaccinated mice (n=5 mice per each challenge dose and strain).Similarly, reactivating virus was not detected when DRGs from ΔgD-2vaccinated mice (isolated Day 5 post-challenge with LD₉₀ of SD90) wereco-cultured for 3 weeks with Vero cells. In contrast, viral DNA andreactivatable virus was recovered from all control (VD60 celllysate)-vaccinated mice (DRG isolated at time of euthanasia) (FIG. 10D,n=5 mice per group).

HSV-2 ΔgD-2 recruits HSV-2 specific IgG2 antibodies and immune cellsinto the skin following challenge: To characterize the immune responseto the vaccine and viral challenge in the skin, biopsies were obtained21 days post boost and 2 or 5 days-post challenge and processed forhistology and/or homogenized and then evaluated for presence ofHSV-specific Abs by ELISA using either an HSV-2(4674) or HSV-1(17)infected cell lysate as the antigen. The ΔgD-2 immunized mice had lowlevels of HSV-specific Abs detected in the skin post-boost, whichrapidly increased as early as day 2 post-challenge (FIG. 11A). B3×1.1elicited higher titer Ab response compared to SD90. As expected, noHSV-specific Abs were detected in the skin of control-vaccinated mice onDay 2 or Day 5 post-challenge (FIGS. 11A and 11B). The Abs recoveredfrom the skin were predominantly IgG [1:24,000 titer, (FIG. 11B)] withno detectable anti-HSV IgA or IgM (data not shown), and were enriched inIgG2 (equal induction of IgG2a and IgG2b) HSV specific antibodies (˜80%of all HSV IgG) (FIG. 11C).

Murine IgG2 antibodies bind FcγR (78). The Abs elicited by the ΔgD-2vaccine mediated ADCC and the studies were extended by measuringantibody-dependent-cellular-phagocytosis (ADCP) against HSV coatedbeads. Serum from ΔgD-2 vaccinated mice obtained 1 week post-boostelicited higher HSV specific phagocytosis and induced greater IFN-γsecretion compared to serum from control immunized mice or beads coatedwith cell lysates (FIG. 11D).

Skin biopsies from ΔgD-2-immunized and control vaccinated mice obtainedon Day 5 post-challenge or unimmunized, uninfected mice (mock) were alsoevaluated by immunohistochemistry and/or immunofluorescence for immunecell responses. The ΔgD-2-immunized mice have a marked increase in CD3+T cells (mean±SD 8.0%±2.1 vs. 2.5%±0.7, p<0.001; FIGS. 12A, 12D) andB220+ B cells (3.8%±1.1 vs; 2.4%±1.1, p=0.09; FIG. 12B, 12E) compared tocontrol immunized mice. The T-cells were further characterized bystaining for CD4 or CD8; there was a significant increase in the CD4+population but not the CD8+ population in ΔgD-2 compared tocontrol-immunized mice (p<0.05) (FIGS. 12F and 12G). Conversely, therewas a decrease in Iba1+ monocyte/macrophage cells (FIGS. 12C and 12H)and Ly6G+ neutrophils (FIG. 13) in ΔgD-2 compared to control immunizedmice.

Consistent with the decrease in inflammatory cells and rapid clearanceof virus, there was a decrease in inflammatory cytokines/chemokinesdetected in skin homogenates in the ΔgD-2 compared to control immunizedmice on Day 5 post-challenge (FIG. 14). HSV-1 and HSV-2-infected micehad higher levels of TNFα (FIG. 14A), IL-1(3 (FIG. 14B), and IL-6 (FIG.14C) compared to mock-infected mice on Day 2 independent ofimmunization. However, the levels decreased by Day 5 in ΔgD-2 but notthe control-immunized mice. Similar results were obtained for thechemokines CXCL9 (FIG. 14D) and CXCL10 (FIG. 14E). Interestingly, IL-33levels were consistently higher in ΔgD-2-immunized compared tocontrol-immunized mice at both time points (FIG. 14F).

Discussion

This study further confirms that vaccination with HSV-2 ΔgD-2 affordscomplete protection against a panel of genetically diverse HSV-1 andHSV-2 clinical isolates and prevents the establishment of latency.Vaccine efficacy was confirmed in an optimized skin model, which isreflective of human primary disease. The ΔgD-2 elicited high titerantibodies that were rapidly recruited into the skin resulting inclearance of virus by day 5 even following challenge with 100-times theLD₉₀ of the most virulent strain, SD90. The protective effect of ΔgD-2against a broad array of HSV-1 and HSV-2 clinical isolatesdifferentiates it from other candidate vaccines such as HSV-2ΔUL5/ΔUL29, which failed to fully protect against the clinical isolateSD90, or gD subunit vaccines and others that have only been testedagainst one or two laboratory viral strains.

The broad protection afforded by ΔgD-2 likely reflects the unique natureof the immune response elicited. The Abs induced were enriched for theIgG2 subtype (˜80% of all HSV-specific IgG), had low level neutralizingactivity (not shown), were rapidly recruited into the skin with titersreaching 1:24,000 in skin biopsies by day 2 post-challenge, and mediatedFc effector functions (78) including ADCP (shown here) and ADCC. Theantibody response to ΔgD-2 was dose-dependent, correlated with therapidity of viral clearance as evidenced by disease scores and likelycontributed to the vaccine's ability to completely prevent theestablishment of latency. Although immunization with lower doses ofΔgD-2 elicited a lower titer of HSV-specific antibody in the serum, allof the mice were protected from lethal challenge. These findings suggestthat lower levels of antibody may be sufficient for FcγR-effectorfunctions. A different HSV-2 viral strain in which the nectin-1 bindingdomain of gD is altered (gD27), also elicited lower titers of serumneutralizing Abs compared to recombinant adjuvanted gD, but, conversely,was more protective than recombinant gD protein against vaginalchallenge in mice (59). Other antibody functions such as ADCC or ADCPwere not evaluated. However, these findings further support the notionthat neutralizing Ab titers are not a predictive correlate of protectionin mice, cotton rats (80), or humans.

While a rapid inflammatory response characterized by increases incytokines and chemokines was observed in both control vaccinated andΔgD-2 vaccinated mice on Day 2 post-challenge, the inflammatory responseresolved in the vaccinated mice by Day 5, which is consistent with therapid clearance of virus. In contrast, inflammation persisted in thecontrol vaccinated mice, consistent with progressive disease. The latterwas characterized by persistently elevated cytokines/chemokines (IL-1β,IL6, CXCL9 and CXCL10) and the persistence of monocytes/macrophages andneutrophils, which were observed throughout the epithelium and withinthe dermal layer in the control-vaccinated mice. In contrast, a higherpercentage of CD4+ T cells and B220+ B cells were observed in the skinof ΔgD-2 vaccinated mice on Day 5, presumably reflecting a cellularmemory response.

Interestingly, IL-33 was the only cytokine that trended higher in theskin from the ΔgD-2 immunized mice compared to controls. The preciserole of IL-33 is not known. Prior studies have shown that rIL-33administration enhanced skin wound healing in mice and was associatedwith activation of innate lymphoid cells and differentiation ofmonocytes into type 2 macrophages (81, 82). Systemic administration ofIL-33 to mice was associated with an increase in FcgR2b, which is linkedto decreased inflammation (88). Possibly, the increase in IL-33 observedin the skin of ΔgD-2 vaccinated mice promoted wound healing andresolution of inflammation.

The clinical isolates displayed variable virulence in the murine skin(and vaginal) model despite similar in vitro growth kinetics (76, 84).Interestingly, however, although a similar level of genetic diversitywas seen among HSV-1 isolates to that described in previous studies,substantially greater genetic diversity was found among the HSV-2isolates collected in the Bronx than those described in previousreports. The greater differences observed here may reflect the diversegeographic origins of the Bronx community. Despite this heterogeneity,all of the isolates tested were completely protected by ΔgD-2 vaccine,possibly reflecting the polyantigenic response. Moreover, completeprotection was observed against both HSV-1 and HSV-2, which isclinically relevant, as HSV-1 has emerged as the more common cause ofgenital disease in the developed world. The universal protectionobserved here combined with the “sterilizing immunity” as evidenced byabsence of latent virus supports the effectiveness of this ΔgD-2vaccine.

Material and Methods

Cells and viruses: Vero (African green monkey kidney cell line; CCL-81;American Type Culture Collection (ATCC), Manassas, Va., USA) cells, VD60cells [Vero cells encoding gD-1 under endogenous promoter (85)], andCaSki (human cervical epithelial cell line; CRL-1550; ATCC) werepassaged in DMEM supplemented with 10% fetal bovine serum (FBS, GeminiBio-Products, West Sacramento, Calif.). THP-1 (human monocyte cell line;TIB-202; ATCC) cells were passaged in RPMI-1640 (Life Technologies)supplemented with 10% FBS and sub-cultured according to ATCC guidelines.Construction of HSV-2(G) ΔgD-2 and its propogation on VD60 cells hasbeen previously described (95, 94). No variability in vaccine efficacyhas been observed comparing 5 different viral preparations. HSV-2(4674)(86) was propagated on CaSki cells. Laboratory strains HSV-2(G) (87),HSV-2 (333-ZAG) (86), HSV-1(17) (89), and HSV-1(F) (87) were propagatedon Vero cells. South African isolate HSV-2(SD90) (97) was provided byDavid Knipe and propagated on Vero cells. Five HSV-1 (B3×1.1 throughB3×1.5) and five HSV-2 (B3×2.1 through B3×2.5) de-identified clinicalisolates were provided by the Clinical Virology Lab at Montefiore andpassaged three times on Vero cells for a low-passage working stock.

In vitro growth curves: Single-step and multi-step growth curves wereperformed as previously described (86). For single-step growth of eachvirus, Vero cells were infected with virus at a multiplicity ofinfection (moi) of 5 PFU/cell and supernatants and cells were collectedevery 4, 8, 16 and 24 hours (h) post-infection (pi) and stored at −80°C. For multi-step growth of each virus, Vero cells were infected at amoi of 0.01 PFU/cell and supernatants and cells were harvested every 12h pi up to 72 hours. Infectious virus was measured by performing plaqueassays with supernatants and lysed cells.

Viral DNA isolation and sequencing of clinical isolates: HSV DNA wasprepared by infecting confluent Vero cells in a T150 flask with each ofthe B3× clinical isolates at an MOT of 10. Cells were harvested 16 hpiand washed twice with PBS. DNA was extracted using DNeasy® Blood andTissue (Qiagen) following the manufacturer's recommendations. DNA wasquantitated by Qubit dsDNA hs assay (Life Technologies). Paired-endlibraries were prepared by the Nextera XT DNA library preparation kit(Illumina) following the manufacturer's instructions. Libraries weresequenced on a Illumina MiSeq Desktop Sequencer. Viral genome sequenceswere assembled with the VirAmp pipeline (89) following removal of hostsequence by alignment to the Macaca mulatta genome as a substitute forthe incomplete Chlorocebus sabaeus (source of Vero cells) genome. HSV-1and HSV-2 genomes were annotated with Genome Annotation Transfer Utilityon ViPR by comparison to HSV-1(96) (GenBank accession no. JN555585.1) &HSV-2(HG52) (JN561323) prior to submission to GenBank. Whole genomealignments including the previously sequenced HSV-2(SD90e) (KF781518),HSV-2(333) (KP192856), ChHV 105640 (NC 023677.1), & HSV-1(F)(GU734771.1) were performed using ClustalW (90) and phylogenetic treeswere constructed using the UPGMA method with 1000 bootstrap replicatesin MEGA6 (91). All positions containing gaps or missing data wereeliminated. GenBank numbers for the genome sequences are as follows:HSV-2(G) (KU310668), HSV-2(4674) (KU310667), B3×1.1 (KU310657), B3×1.2(KU310658), B3×1.3 (KU310659), B3×1.4 (KU310660), B3×1.5 (KU310661),B3×2.1 (KU310662), B3×2.2 (KU310663), B3×2.3 (KU310664), B3×2.4(KU310665), B3×2.5 (KU310666).

Murine immunization and viral challenge studies: Experiments wereperformed with approval from Albert Einstein College of MedicineInstitutional Animal Care and Use Committee, Protocol #20130913 and#20150805. Female C57BL/6 and BALB/c mice were purchased from JacksonLaboratory (JAX, Bar Harbor, Me.) at 4-6 weeks of age. Mice were primedand boosted 3 weeks later with 5×10⁴-5×10⁶ PFU of ΔgD-2 or equal amountof VD60 cell lysates (Control) subcutaneously (sc, medial to the hindlimb and pelvis) at 100 μl/mouse. The titer was determined by a plaqueassay on complementing cells (VD60).

For intravaginal HSV infections, mice were treated with 2.5 mg ofmedoxyprogesterone acetate (MPA; Sicor Pharmaceuticals, Irvine, Calif.)sc five days prior to challenge. Mice were then inoculatedintravaginally with an LD90 (5×10^5 pfu/mouse) of HSV-2(4674) at 30μl/mouse and scored for disease and monitored for survival for 14 daysas previously described (21). For HSV skin infections, mice weredepilated on the right flank with Nair and allowed to rest for 24 hr.Depilated mice were anesthetized with isoflurane (Isothesia,Henry-Schein), then abraded on the exposed skin with a disposable emoryboard for 20-25 strokes and subsequently challenged with 1×10⁵ PFU HSV-1or 5×10⁴ PFU HSV-2 strains for in vivo virulence studies or challengedwith an LD₉₀, 10×LD₉₀, or 100×LD₉₀ of select HSV strains (see Table 1)for vaccine efficacy studies. Mice were monitored for 14 days and scoredas follows: 1: primary lesion or erythema, 2: distant site zosteriformlesions, mild edema/erythema, 3: severe ulceration and edema, increasedepidermal spread, 4: hind-limp paresis/paralysis and 5: death. Mice thatwere euthanized at a score of 4 were assigned a value of 5 on allsubsequent days for statistical analyses.

HSV RT-qPCR: DNA was extracted from weighed tissue samples using DNeasy®Blood and Tissue (Qiagen) following the manufacturer's recommendations.Extracted DNA was then normalized to 10 ng of DNA per reaction and viralDNA quantified using real-time quantitative PCR (RT-qPCR, qPCR) usingABsolute qPCR ROX Mix (Thermo Scientific). Primers for HSV polymerase(UL30) were purchased from Integrated DNA Technologies (Cat#:1179200494) and used to detect viral genomic DNA. Isolated HSV-2 viralDNA was calibrated for absolute copy amounts using QuantStudio® 3DDigital PCR (dPCR, ThermoFisher Scientific) and subsequently used as astandard curve to determine HSV viral genome copies. Samples that read 4or less copy numbers were considered negative. Data are presented as log10 HSV genomes per gram of DRG (dorsal root ganglia) tissue.

Detection of antibodies and cytokines in skin biopsies: Skin biopsieswere obtained from HSV-2 ΔgD-2 or VD60 lysates (control) immunized mice(˜5-10 mm in diameter by mechanical excision) day 21 post-boost or day 2and 5 post viral skin challenge. The tissue was weighed and homogenizedin RNase/DNase free Lysing Matrix A tubes (MP Biomedicals, Santa Ana,Calif.) with serum-free DMEM at 6.0 m/sec for three 30 sec cycles in theFastPrep-24™ 5G (MP Biomedicals). Samples were spun at 5000 rpm for 10min at 4° C. and the resulting supernatant was evaluated for anti-HSVantibodies, cytokines and chemokines. Anti-HSV antibodies were detectedby ELISA as previously described using uninfected, HSV-1(96), orHSV-2(4674)-infected Vero cell lysates as the coating antigen (94).Biotin anti-mouse Ig κ or biotin anti-mouse IgA, IgM, IgG1, IgG2a,IgG2b, or IgG3 at 1 μg/ml (Becton Dickenson, San Diego) were used assecondary detection antibodies. Wells were read on a SpectraMax (M5series) ELISA plate reader at an absorbance of 450 nm. The resultingabsorbance was determined by subtracting values obtained for uninfectedcell lysates to values obtained with infected cell lysates. Totalanti-HSV Ig is reported as the optical density (OD) at 450 nm normalizedto relative tissue weight at a 1:1000 dilution of tissue homogenate.Anti-HSV IgG, IgA, IgM, or IgG1-3 are reported as the optical density(OD) at 450 nm at all dilutions except IgG1-3 which is reported only ata 1:100 dilution of skin homogenate.

Skin homogenate supernatants were assayed for interleukin-6 (IL-6), IL-1beta (IL-1β), IL-33, tumor necrosis factor alpha (TNFα), monokineinduced by interferon-gamma (MIG, CXCL9), interferon-inducible cytokine(IP-10, CXCL10) using a Milliplex mouse cytokine/chemokine immunoassay(Millipore, Danvers, Mass.) and a Luminex Magpix system and analyzedwith Milliplex Analyst (Version 3.5.5.0; VigeneTech Inc.).

Histopathology, immunohistochemistry and immunofluorescence of skintissue: Mice were euthanized on Day 5 post-challenge and the skin at theviral (or mock) infection site was excised and formalin fixed for 48 hrsat RT. Samples were processed routinely to be paraffin-embedded andsectioned. Slides for histopathology were stained with hematoxylin andeosin (H&E). Samples were evaluated histologically by a board certifiedveterinary pathologist which were blinded of samples identity. Forimmunohistochemistry (IHC), the samples were sectioned to 5 μm,deparaffinized in xylene followed by graded alcohols. Antigen retrievalwas performed in 10 mM sodium citrate buffer at pH 6.0, heated to 96°C., for 30 minutes. Endogenous peroxidase activity was blocked using 3%hydrogen peroxide in water. The sections were stained by routine IHCmethods, using SuperPicTure™ (ThermoFisher Scientific, Cat:87-9673)against rabbit primary antibodies to anti-CD3 (Ready to use format,ThermoFisher Scientific, Cat: RM-9107-R7), anti-B220 (BD BiosciencesCat: 550286), or anti-Iba1 (1:3000 dilution Wako Pure ChemicalIndustries, Richmond, Va.) and then stained with diaminobenzidine as thefinal chromogen. All immunostained sections were lightly counterstainedwith hematoxylin. Stained cross-sections were photomicrographed withZeiss Axio Observer inverted light microscope at 20× magnification fromapical layer (epidermal) to basal layer (striated muscle) at 3 differentlocations per sample. Stained-positive cells were enumerated asdescribed by Bologna-Molina et al., 2011 (92). Data is represented asthe average of % positive cells=(positive nucleated cells/totalnucleated cells) of three photomicrographed sections per sample.

For Immunofluorescent studies, skin tissue was excised 5 days post-HSVor mock skin challenge and then frozen in OCT media. Samples were cutinto 5 μm sections and stored at −80° C. Frozen slides were then fixedin −20° C. acetone for 15 mins, washed with wash buffer (WB, 0.05% Tween20 in PBS), then blocked for 2 hrs with blocking buffer (2% BSA, 5% heatinactivated goat serum in PBS) at RT. Slides were washed twice andincubated with anti-CD4(GK1.5, 1:200), anti-CD8 (YTS 169.4, 1:250),anti-Ly6G (1A8, 1:500) in blocking buffer for 1 hr at RT. Slides werethoroughly washed and incubated with an goat anti-rat secondary antibodyconjugated with either Alexa flour 555 or Alexa flour 488 (1:500 or1:200, respectively) for 30 min at RT. Slides were washed and mountedwith media containing DAPI (ProLong® Diamond Antifade Mountant withDAPI, ThermoFisher Scientific). Slides were imaged using a Nikon EclipseTi-U inverted light microscope at 20× magnification from apical layer(epidermal) to basal layer (striated muscle) at two different locationsper sample. For % CD4+ and % CD8+ quantification, total nucleated cellswere calculated by DAPI positive objects ≥5 μm via a software algorithmfrom Velocity (version 6.3, PerkinElmer). CD4 or CD8 positive cells werecounted manually for fluorescence and incorporation of a DAPI+ nuclei toexclude non-specific staining of hair follicles and cellular debris inthe skin sections. Data is represented as the average of % positivecells=(positive cells/total DAPI cells) of two images per sample.

Antibody dependent cellular phagocytosis (ADCP) assay. To determine HSVspecific ADCP, a protocol modified from Ackerman et al., 2011 (93) wasused. Briefly, 2×108 1 μm Neutravadin-red fluorescent beads (Invitrogen,F-8775) were coated with 0.3 mg of biotinylated HSV-2 infected oruninfected (control) Vero cells overnight at 4° C. in 500 μl ofBlockAid™ (ThermoFisher Scientific, B-10710). Beads were washed twicewith 1% BSA in PBS and then 1×10⁶ beads/well were added in a 96 roundbottom plate. Serum from immunized mice at 1 week post boost washeat-inactivated at 56° C. for 30 min and diluted 1:5 in serum-freeRPMI. 50 μl of diluted serum was added to wells that contained the HSVlysates or control cell lysates coated beads and incubated for 2 h at37° C. 2×10⁴ cells/well THP-1 cells were added to each at a final volumeof 200 μl/well and incubated for 8 hr at 37° C. at 5% CO₂. Subsequently,100 μl of supernatant was removed and stored at −20° C. then resuspendedwith 100 μl 4% paraformaldehyde. Samples were then read on 5-laser LSRIIflow cytometer (Becton Dickenson, San Diego) at the Einstein FlowCytometry Core Facility. Phagocytic score is reported by gating onevents representing THP-1 cells then applying the following equation:[(% of cells bead positive X MFI of cells positive for beads)/10⁶] usingFlowJo software (version 10, Tree Star Inc.). IFN-γ secretion fromactivated THP-1 cells via antibody phagocytosis was determined byanalyzing stored cultured supernatants using a Milliplex human customimmunoassay (Millipore, Danvers, Mass.) and a Luminex Magpix system aspreviously described.

Virus detection in tissue. Skin and dorsal root ganglia (DRG) wereweighed and homogenized as described above. Supernatants of homogenizedtissue were then overlaid on confluent Vero cell monolayers (2×105cells/well in a 48-well plate) for 1 h. Wells were washed with PBS andthen with 199 medium (Gibco®) containing 1% heat-inactivated FBS,overlaid with 0.5% methylcellulose and incubated at 37° C. for 48 h.Cells were fixed with 2% paraformaldehyde, stained with a crystal violetsolution and the number of PFU quantified. Neuronal ex-vivo co-cultureassays were performed as previously described (94).

Statistical analysis. Results were compared by two-way analysis ofvariance (2-way ANOVA) with multiple comparisons or unpaired student'st-tests using GraphPad Prism version 6 (San Diego, Calif.). Mantel-Coxsurvival curves were compared by log rank tests. P values <0.05 (*),<0.01 (**), <0.001 (***) were considered significant.

REFERENCES

-   1. Looker, K. J., G. P. Garnett, and G. P. Schmid, An estimate of    the global prevalence and incidence of herpes simplex virus type 2    infection. Bull World Health Organ, 2008. 86(10): p. 805-12, A.-   2. Freeman, E. E., et al., Herpes simplex virus 2 infection    increases HIV acquisition in men and women: systematic review and    meta-analysis of longitudinal studies. AIDS, 2006. 20(1): p. 73-83.-   3. Gray, R. H., et al., Probability of HIV-1 transmission per coital    act in monogamous, heterosexual, HIV-1-discordant couples in Rakai,    Uganda. Lancet, 2001. 357(9263): p. 1149-53.-   4. Wald, A. and K. Link, Risk of human immunodeficiency virus    infection in herpes simplex virus type 2-seropositive persons: a    meta-analysis. J Infect Dis, 2002. 185(1): p. 45-52.-   5. Paz-Bailey, G., et al., Herpes simplex virus type 2: epidemiology    and management options in developing countries. Sex Transm    Infect, 2007. 83(1): p. 16-22.-   6. Doi, Y., et al., Seroprevalence of herpes simplex virus 1 and 2    in a population-based cohort in Japan. J Epidemiol, 2009. 19(2): p.    56-62.-   7. Bradley, H., et al., Seroprevalence of herpes simplex virus types    1 and 2—United States, 1999-2010. J Infect Dis, 2014. 209(3): p.    325-33.-   8. Belshe, R. B., et al., Efficacy results of a trial of a herpes    simplex vaccine. N Engl J Med, 2012. 366(1): p. 34-43.-   9. Bernstein, D. I., et al., Epidemiology, clinical presentation,    and antibody response to primary infection with herpes simplex virus    type 1 and type 2 in young women. Clin Infect Dis, 2013. 56(3): p.    344-51.-   10. Kimberlin, D., Herpes simplex virus, meningitis and encephalitis    in neonates. Herpes, 2004. 11 Suppl 2: p. 65A-76A.-   11. Ward, K. N., et al., Herpes simplex serious neurological disease    in young children: incidence and long-term outcome. Arch Dis    Child, 2012. 97(2): p. 162-5.-   12. Lafferty, W. E., et al., Recurrences after oral and genital    herpes simplex virus infection. Influence of site of infection and    viral type. N Engl J Med, 1987. 316(23): p. 1444-9.-   13. Owusu-Edusei, K., Jr., et al., The estimated direct medical cost    of selected sexually transmitted infections in the United    States, 2008. Sex Transm Dis, 2013. 40(3): p. 197-201.-   14. Mertz, G. J., et al., Double-blind, placebo-controlled trial of    a herpes simplex virus type 2 glycoprotein vaccine in persons at    high risk for genital herpes infection. J Infect Dis, 1990.    161(4): p. 653-60.-   15. Group, H. S. V. S., et al., Safety and immunogenicity of a    glycoprotein D genital herpes vaccine in healthy girls 10-17 years    of age: results from a randomised, controlled, double-blind trial.    Vaccine, 2013. 31(51): p. 6136-43.-   16. Leroux-Roels, G., et al., Immunogenicity and safety of different    formulations of an adjuvanted glycoprotein D genital herpes vaccine    in healthy adults: a double-blind randomized trial. Hum Vaccin    Immunother, 2013. 9(6): p. 1254-62.-   17. Bernstein, D. I., et al., Safety and immunogenicity of    glycoprotein D-adjuvant genital herpes vaccine. Clin Infect    Dis, 2005. 40(9): p. 1271-81.-   18. Stanberry, L. R., et al., Glycoprotein-D-adjuvant vaccine to    prevent genital herpes. N Engl J Med, 2002. 347(21): p. 1652-61.-   19. Corey, L., et al., Recombinant glycoprotein vaccine for the    prevention of genital HSV-2 infection: two randomized controlled    trials. Chiron HSV Vaccine Study Group. JAMA, 1999. 282(4): p.    331-40.-   20. jh.richardus@rotterdam.nl, Safety and immunogenicity of a    glycoprotein D genital herpes vaccine in healthy girls 10-17 years    of age: Results from a randomised, controlled, double-blind trial.    Vaccine, 2013. 31(51): p. 6136-43.-   21. Belshe, R. B., et al., Correlate of Immune Protection Against    HSV-1 Genital Disease in Vaccinated Women. J Infect Dis, 2013.-   22. Gerber, S. I., B. J. Belval, and B. C. Herold, Differences in    the role of glycoprotein C of HSV-1 and HSV-2 in viral binding may    contribute to serotype differences in cell tropism. Virology, 1995.    214(1): p. 29-39.-   23. Lubinski, J. M., et al., The herpes simplex virus 1 IgG fc    receptor blocks antibody-mediated complement activation and    antibody-dependent cellular cytotoxicity in vivo. J Virol, 2011.    85(7): p. 3239-49.-   24. Para, M. F., L. Goldstein, and P. G. Spear, Similarities and    differences in the Fc-binding glycoprotein (gE) of herpes simplex    virus types 1 and 2 and tentative mapping of the viral gene for this    glycoprotein. J Virol, 1982. 41(1): p. 137-44.-   25. Hook, L. M., et al., Herpes simplex virus type 1 and 2    glycoprotein C prevents complement-mediated neutralization induced    by natural immunoglobulin M antibody. J Virol, 2006. 80(8): p.    4038-46.-   26. Lubinski, J. M., et al., Herpes simplex virus type 1 evades the    effects of antibody and complement in vivo. J Virol, 2002.    76(18): p. 9232-41.-   27. Awasthi, S., et al., Immunization with a vaccine combining    herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits    improves the protection of dorsal root ganglia in mice and reduces    the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea    pigs compared to immunization with gD alone. J Virol, 2011.    85(20): p. 10472-86.-   28. Manservigi, R., et al., Immunotherapeutic activity of a    recombinant combined gB-gD-gE vaccine against recurrent HSV-2    infections in a guinea pig model. Vaccine, 2005. 23(7): p. 865-72.-   29. de Bruyn, G., et al., A randomized controlled trial of a    replication defective (gH deletion) herpes simplex virus vaccine for    the treatment of recurrent genital herpes among immunocompetent    subjects. Vaccine, 2006. 24(7): p. 914-20.-   30. Ouwendijk, W. J., et al., T-cell immunity to human    alphaherpesviruses. Curr Opin Virol, 2013. 3(4): p. 452-60.-   31. Parr, M. B. and E. L. Parr, Mucosal immunity to herpes simplex    virus type 2 infection in the mouse vagina is impaired by in vivo    depletion of T lymphocytes. J Virol, 1998. 72(4): p. 2677-85.-   32. Noisakran, S. and D. J. Carr, Lymphocytes delay kinetics of    HSV-1 reactivation from in vitro explants of latent infected    trigeminal ganglia. J Neuroimmunol, 1999. 95(1-2): p. 126-35.-   33. van Velzen, M., et al., Local CD4 and CD8 T-cell reactivity to    HSV-1 antigens documents broad viral protein expression and immune    competence in latently infected human trigeminal ganglia. PLoS    Pathog, 2013. 9(8): p. e1003547.-   34. Muller, W. J., et al., Herpes simplex virus type 2 tegument    proteins contain subdominant T-cell epitopes detectable in BALB/c    mice after DNA immunization and infection. J Gen Virol, 2009. 90(Pt    5): p. 1153-63.-   35. Zhu, J., et al., Immune surveillance by CD8alphaalpha+    skin-resident T cells in human herpes virus infection. Nature, 2013.    497(7450): p. 494-7.-   36. Steinberg, M. W., et al., Regulating the mucosal immune system:    the contrasting roles of LIGHT, HVEM, and their various partners.    Semin Immunopathol, 2009. 31(2): p. 207-21.-   37. Steinberg, M. W., T. C. Cheung, and C. F. Ware, The signaling    networks of the herpesvirus entry mediator (TNFRSF14) in immune    regulation. Immunol Rev, 2011. 244(1): p. 169-87.-   38. Kopp, S. J., C. S. Storti, and W. J. Muller, Herpes simplex    virus-2 glycoprotein interaction with HVEM influences virus-specific    recall cellular responses at the mucosa. Clin Dev Immunol, 2012.    2012: p. 284104.-   39. Yoon, M., et al., Functional interaction between herpes simplex    virus type 2 gD and HVEM transiently dampens local chemokine    production after murine mucosal infection. PLoS One, 2011. 6(1): p.    e16122.-   40. Ligas, M. W. and D. C. Johnson, A herpes simplex virus mutant in    which glycoprotein D sequences are replaced by beta-galactosidase    sequences binds to but is unable to penetrate into cells. J    Virol, 1988. 62(5): p. 1486-94.-   41. Cheshenko, N., et al., HSV activates Akt to trigger calcium    release and promote viral entry: novel candidate target for    treatment and suppression. FASEB J, 2013. 27(7): p. 2584-99.-   42. Parr, E. L. and M. B. Parr, Immunoglobulin G is the main    protective antibody in mouse vaginal secretions after vaginal    immunization with attenuated herpes simplex virus type 2. J    Virol, 1997. 71(11): p. 8109-15.-   43. Mbopi-Keou, F. X., et al., Cervicovaginal neutralizing    antibodies to herpes simplex virus (HSV) in women seropositive for    HSV Types 1 and 2. Clin Diagn Lab Immunol, 2003. 10(3): p. 388-93.-   44. Hendrickson, B. A., et al., Decreased vaginal disease in    J-chain-deficient mice following herpes simplex type 2 genital    infection. Virology, 2000. 271(1): p. 155-62.-   45. Nixon, B., et al., Genital Herpes Simplex Virus Type 2 Infection    in Humanized HIV-Transgenic Mice Triggers HIV Shedding and Is    Associated With Greater Neurological Disease. J Infect Dis, 2013.-   46. Carr, D. J. and L. Tomanek, Herpes simplex virus and the    chemokines that mediate the inflammation. Curr Top Microbiol    Immunol, 2006. 303: p. 47-65.-   47. Stefanidou, M., et al., Herpes simplex virus 2 (HSV-2) prevents    dendritic cell maturation, induces apoptosis, and triggers release    of proinflammatory cytokines: potential links to HSV-HIV synergy. J    Virol, 2013. 87(3): p. 1443-53.-   48. Bourne, N., et al., Herpes simplex virus (HSV) type 2    glycoprotein D subunit vaccines and protection against genital HSV-1    or HSV-2 disease in guinea pigs. J Infect Dis, 2003. 187(4): p.    542-9.-   49. Bourne, N., et al., Impact of immunization with glycoprotein    D2/ASO4 on herpes simplex virus type 2 shedding into the genital    tract in guinea pigs that become infected. J Infect Dis, 2005.    192(12): p. 2117-23.-   50. Bernstein, D. I., et al., The adjuvant CLDC increases protection    of a herpes simplex type 2 glycoprotein D vaccine in guinea pigs.    Vaccine, 2010. 28(21): p. 3748-53.-   51. Bernstein, D. I., et al., Potent adjuvant activity of cationic    liposome-DNA complexes for genital herpes vaccines. Clin Vaccine    Immunol, 2009. 16(5): p. 699-705.-   52. Sweeney, K. A., et al., A recombinant Mycobacterium smegmatis    induces potent bactericidal immunity against Mycobacterium    tuberculosis. Nat Med, 2011. 17(10): p. 1261-8.-   53. Kohl, S., et al., Limited antibody-dependent cellular    cytotoxicity antibody response induced by a herpes simplex virus    type 2 subunit vaccine. J Infect Dis, 2000. 181(1): p. 335-9.-   54. John, M., et al., Cervicovaginal secretions contribute to innate    resistance to herpes simplex virus infection. J Infect Dis, 2005.    192(10): p. 1731-40.-   55. Nugent, C. T., et al., Analysis of the cytolytic T-lymphocyte    response to herpes simplex virus type 1 glycoprotein B during    primary and secondary infection. J Virol, 1994. 68(11): p. 7644-8.-   56. Mueller, S. N., et al., Characterization of two TCR transgenic    mouse lines specific for herpes simplex virus. Immunol Cell    Biol, 2002. 80(2): p. 156-63.-   57. Wallace, M. E., et al., The cytotoxic T-cell response to herpes    simplex virus type 1 infection of C57BL/6 mice is almost entirely    directed against a single immunodominant determinant. J Virol, 1999.    73(9): p. 7619-26.-   58. Milligan, G. N., et al., T-cell-mediated mechanisms involved in    resolution of genital herpes simplex virus type 2 (HSV-2) infection    of mice. J Reprod Immunol, 2004. 61(2): p. 115-27.-   59. Wang, K., et al., A herpes simplex virus 2 glycoprotein D mutant    generated by bacterial artificial chromosome mutagenesis is severely    impaired for infecting neuronal cells and infects only Vero cells    expressing exogenous HVEM. J Virol, 2012. 86(23): p. 12891-902.-   60. Barletta, R. G., et al., Identification of expression signals of    the mycobacteriophages Bxb1, L1 and TM4 using the    Escherichia-Mycobacterium shuttle plasmids pYUB75 and pYUB76    designed to create translational fusions to the lacZ gene. J Gen    Microbiol, 1992. 138(1): p. 23-30.-   61. Yamaguchi, S., et al., A method for producing transgenic cells    using a multi-integrase system on a human artificial chromosome    vector. PLoS One, 2011. 6(2): p. e17267.-   62. Xu, Z., et al., Accuracy and efficiency define Bxb1 integrase as    the best of fifteen candidate serine recombinases for the    integration of DNA into the human genome. BMC Biotechnol, 2013.    13: p. 87.-   63. Hill, A., et al., Herpes simplex virus turns off the TAP to    evade host immunity. Nature, 1995. 375(6530): p. 411-5.-   64. Shu, M., et al., Selective degradation of mRNAs by the HSV host    shutoff RNase is regulated by the UL47 tegument protein. Proc Natl    Acad Sci USA, 2013. 110(18): p. E1669-75.-   65. Umbach, J. L., et al., MicroRNAs expressed by herpes simplex    virus 1 during latent infection regulate viral mRNAs. Nature, 2008.    454(7205): p. 780-3.-   66. Cheshenko, N., et al., Herpes simplex virus triggers activation    of calcium-signaling pathways. J Cell Biol, 2003. 163(2): p. 283-93.-   67. Cheshenko, N. and B. C. Herold, Glycoprotein B plays a    predominant role in mediating herpes simplex virus type 2 attachment    and is required for entry and cell-to-cell spread. J Gen    Virol, 2002. 83(Pt 9): p. 2247-55.-   68. Cheshenko, N., et al., Multiple receptor interactions trigger    release of membrane and intracellular calcium stores critical for    herpes simplex virus entry. Mol Biol Cell, 2007. 18(8): p. 3119-30.-   69. Immergluck, L. C., et al., Viral and cellular requirements for    entry of herpes simplex virus type 1 into primary neuronal cells. J    Gen Virol, 1998. 79 (Pt 3): p. 549-59.-   70. Nixon, B., et al., Genital Herpes Simplex Virus Type 2 Infection    in Humanized HIV-Transgenic Mice Triggers HIV Shedding and Is    Associated With Greater Neurological Disease. J Infect Dis, 2014.    209(4): p. 510-22.-   71. Cheshenko, N., et al., HSV usurps eukaryotic initiation factor 3    subunit M for viral protein translation: novel prevention target.    PLoS One, 2010. 5(7): p. e11829.-   72. Carbonetti, S., et al., Soluble HIV-1 Envelope Immunogens    Derived from an Elite Neutralizer Elicit Cross-Reactive V1V2    Antibodies and Low Potency Neutralizing Antibodies. PLoS One, 2014.    9(1): p. e86905.-   73. Janes, H., et al., Vaccine-induced gag-specific T cells are    associated with reduced viremia after HIV-1 infection. J Infect    Dis, 2013. 208(8): p. 1231-9.-   74. Ferre, A. L., et al., Immunodominant HIV-specific CD8+ T-cell    responses are common to blood and gastrointestinal mucosa, and    Gag-specific responses dominate in rectal mucosa of HIV controllers.    J Virol, 2010. 84(19): p. 10354-65.-   75. Schiffer J T, Corey L (2013) Rapid host immune response and    viral dynamics in herpes simplex virus-2 infection. Nat Med    19:280-90.-   76. Sydiskis, Schultz (1965) Herpes simplex skin infection in mice.    J Infect Dis 115:237-46.-   77. Nixon B et al. (2013) Griffithsin protects mice from genital    herpes by preventing cell-to-cell spread. J Virol 87:6257-69.-   78. Nimmerjahn F, Bruhns P, Horiuchi K, Ravetch J V (2005)    FcgammaRlV: a novel FcR with distinct IgG subclass specificity.    Immunity 23:41-51.-   79. Wang K et al. (2015) A Herpes Simplex Virus 2 (HSV-2) gD Mutant    Impaired for Neural Tropism Is Superior to an HSV-2 gD Subunit    Vaccine To Protect Animals from Challenge with HSV-2. J Virol    90:562-74.-   80. Boukhvalova M et al. (2015) Efficacy of the Herpes Simplex Virus    2 (HSV-2) Glycoprotein D/ASO4 Vaccine against Genital HSV-2 and    HSV-1 Infection and Disease in the Cotton Rat Sigmodon hispidus    Model. J Virol 89:9825-40.-   81. Yin H et al. (2013) IL-33 accelerates cutaneous wound healing    involved in upregulation of alternatively activated macrophages. Mol    Immunol 56:347-53.-   82. Rak G D et al. (2015) IL-33-Dependent Group 2 Innate Lymphoid    Cells Promote Cutaneous Wound Healing. J Invest Dermatol.-   83. Anthony R M, Kobayashi T, Wermeling F, Ravetch J V (2011)    Intravenous gammaglobulin suppresses inflammation through a novel    T(H)2 pathway. Nature 475:110-3.-   84. Simmons, Nash (1984) Zosteriform spread of herpes simplex virus    as a model of recrudescence and its use to investigate the role of    immune cells in prevention of recurrent disease.-   85. Ligas, Johnson (1988) A herpes simplex virus mutant in which    glycoprotein D sequences are replaced by beta-galactosidase    sequences binds to but is unable to penetrate into cells.-   86. Nixon B et al. (2013) Griffithsin protects mice from genital    herpes by preventing cell-to-cell spread. Journal of virology    87:6257-69.-   87. Ejercito, Kieff, Roizman (1968) Characterization of herpes    simplex virus strains differing in their effects on social behaviour    of infected cells. J Gen Virology 2:357-64.-   88. Brown, Ritchie, Subak-Sharpe (1973) Genetic studies with herpes    simplex virus type 1. The isolation of temperature-sensitive    mutants, their arrangement into complementation groups and    recombination analysis leading to a linkage map. The Journal of    general virology 18:329-46.-   89. Wan Y, Renner D W, Albert I, Szpara M L (2015) VirAmp: a    galaxy-based viral genome assembly pipeline. Gigascience 4:19.-   90. Larkin M A et al. (2007) Clustal W and Clustal X version 2.0.    Bioinformatics 23:2947-8.-   91. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013)    MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol    Biol Evol 30:2725-9.-   92. Bologna-Molina R, Damian-Matsumura P, Molina-Frechero N (2011)    An easy cell counting method for immunohistochemistry that does not    use an image analysis program. Histopathology 59:801-3.-   93. Ackerman M et al. (2011) A robust, high-throughput assay to    determine the phagocytic activity of clinical antibody samples.    Journal of Immunological Methods 366:8-19.-   94. Petro C et al. (2015) Herpes simplex type 2 virus deleted in    glycoprotein D protects against vaginal, skin and neural disease.    Elife 4.-   95. Cheshenko N et al. (2013) HSV activates Akt to trigger calcium    release and promote viral entry: novel candidate target for    treatment and suppression. FASEB J 27:2584-99.-   96. Kolb A W, Larsen I V, Cuellar J A, Brandt C R (2015) Genomic,    phylogenetic, and recombinational characterization of herpes simplex    virus 2 strains. J Virol 89:6427-34.-   97. Dudek T E, Torres-Lopez E, Crumpacker C, Knipe D M (2011)    Evidence for differences in immunologic and pathogenesis properties    of herpes simplex virus 2 strains from the United States and South    Africa. J Infect Dis 203:1434-41.

What is claimed is:
 1. A method of eliciting an immune response in asubject comprising administering to the subject a recombinant herpessimplex virus-2 (HSV-2) in an amount effective to elicit an immuneresponse in a subject, wherein said HSV-2 has a deletion of the entireHSV-2 glycoprotein D-encoding gene in the genome thereof; and whereinsaid HSV-2 is phenotypically complemented with a herpes simplex virus-1(HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cellexpressing said HSV-1 glycoprotein D.
 2. A method of treating an HSV-2infection in a subject or treating a disease caused by an HSV-2infection in a subject comprising administering to the subject arecombinant herpes simplex virus-2 (HSV-2) in an amount effective totreat HSV-2 infection and/or HSV-2 disease in a subject, wherein saidHSV-2 has a deletion of the entire HSV-2 glycoprotein D-encoding gene inthe genome thereof; wherein said HSV-2 is phenotypically complementedwith a herpes simplex virus-1 (HSV-1) glycoprotein D by propagating saidHSV-2 in a complementing cell expressing said HSV-1 glycoprotein D; andwherein the recombinant HSV-2, when generated from a non-complementingcell, is non-infectious.
 3. The method of claim 2, wherein the diseasecaused by an HSV-2 infection comprises a genital ulcer.
 4. The method ofclaim 2, wherein the disease caused by an HSV-2 infection comprises askin vesicle or skin ulcer.
 5. A method of vaccinating a subject againstHSV-2 infection comprising administering to the subject an effectiveamount of a composition comprising a recombinant herpes simplex virus-2(HSV-2), wherein said HSV-2 has a deletion of the entire HSV-2glycoprotein D-encoding gene in the genome thereof; wherein said HSV-2is phenotypically complemented with a herpes simplex virus-1 (HSV-1)glycoprotein D by propagating said HSV-2 in a complementing cellexpressing said HSV-1 glycoprotein D; and wherein the recombinant HSV-2,when generated from a non-complementing cell, is non-infectious.
 6. Amethod of inducing antibody dependent cell mediated cytotoxicity (ADCC)and/or antibody-dependent-cellular-phagocytosis (ADCP) against anantigenic target in a subject comprising administering to the subject arecombinant herpes simplex virus-2 (HSV-2) comprising a heterogenousantigen on a lipid bilayer thereof, in an amount effective to induceADCC and/or ADCP against the antigenic target in a subject, wherein saidHSV-2 has a deletion of the entire HSV-2 glycoprotein D-encoding gene inthe genome thereof; wherein said HSV-2 is phenotypically complementedwith a herpes simplex virus-1 (HSV-1) glycoprotein D by propagating saidHSV-2 in a complementing cell expressing said HSV-1 glycoprotein D; andwherein the recombinant HSV-2, when generated from a non-complementingcell, is non-infectious.
 7. The method of claim 5, wherein therecombinant HSV-2 has been produced by a method comprising infecting acell comprising a heterologous nucleic acid encoding a HSV-1glycoprotein D, and expressing HSV-1 glycoprotein D on a cell membranethereof, with a recombinant herpes simplex virus-2 (HSV-2) having adeletion of an entire HSV-2 glycoprotein D-encoding gene in the genomethereof, under conditions permitting replication of the recombinantherpes simplex virus-2 (HSV-2) and recovering recombinant HSV-2 virionscomprising an HSV-1 glycoprotein D on a lipid bilayer thereof producedby the cell.
 8. The method of claim 5, wherein the composition furthercomprises an immunological adjuvant.
 9. The method of claim 5, whereinthe subject is not infected with HSV-2.
 10. The method of claim 9,wherein the composition further comprises an immunological adjuvant. 11.The method of claim 2, 5 or 6, wherein the non-complementing cell is aVero cell.
 12. The method of claim 2, 5 or 6, wherein the complementingcell is a VD60 cell.